DE102004025744A1 - Surface of a solid support, useful for multiple parallel analysis of nucleic acids by optical methods, having low non-specific binding of labeled components - Google Patents

Abstract

Surface (A) of a solid support (B) for parallel analysis of many individual nucleic acids (NA) by optical methods. Independent claims are also included for the following: (1) solid phase, as above, in which the surface shows reduced non-specific binding of labeled components; (2) methods for preparing (B); and (3) methods of parallel analysis of many NA by optical methods, using (B).

Description

The Invention describes a surface a solid phase for a method for sequencing nucleic acid chains.

since In the mid-nineties, procedures are developed, the events on individual molecules or analyze on small groups of individual molecules. In many Procedures become single molecules while the analysis bound to a solid phase. For detection Reaction components labeled, for example, with fluorescence marker. Because the signal from single molecules is usually very weak is, need such Processes including surfaces of solid phases, the generate a low background signal.

The Background signal, e.g. Fluorescence signal, may be from the solid phase originate from marked components, which by the Absorption (e.g., nonspecific absorption) to the solid phase during Reaction were bound.

For the analyzes, on a single molecule basis It is essential that the background signal from the solid phase as possible is low. In particular, the reduction of non-specific Binding of labeled components during the analysis is a challenge represents.

Generally known procedures for the preparation of surfaces for the preparation of biosensors (Ligler, 1999) are mostly ineffective in preparing surfaces for processes, based on single-molecule analysis, as they create a strong background due to non-specific binding lead from marked components.

since In the mid-80s, methods are developed in which nucleic acids are initially on be fixed to a solid phase, followed by a biochemical Reaction, for example hybridization or primer extension, analyzed to become. It was found that the cleaning procedures and the fixation of nucleic acids to the surfaces a big Role play and a clear prediction of the outcome of a Cleaning procedure with regard to potential emergence of the background signal by unspecific binding of labeled components is not possible (Taylor et al., 2003).

In spite of intensive search for suitable surfaces is not yet managed to get a surface too find that despite longer and repeated contact with high concentrations of labeled reagents, as labeled nucleic acids and / or labeled nucleotides, a low background signal having non-specific binding of labeled components.

Object of the invention:

The The object of the invention was to provide a surface of a solid phase, used in the analysis of nucleic acid chains can and is a low non-specific binding of both labeled and unlabelled nucleotides as well as labeled and unlabelled nucleic acids having.

A Another object of the invention was to provide a surface of a solid phase, which has a low non-specific binding of labeled and unlabelled nucleotides and labeled and unlabelled nucleic acids having, to this surface the solid phase nucleic acids, e.g. nucleic acid chains with a length from 10 to 1000 nucleotides, can be coupled.

A Another object of the invention was to develop a surface which can be used in an analysis process in which marked and / or unlabelled nucleotides and / or labeled and / or unlabelled nucleic acids used, for example a sequencing method (WO 02088382, WO 03020968, WO 02072892, WO 0070073, WO 03020968) or also in US Pat DNA chip technology like in ("DNA Microarrays", Bowtell, 2003, ISBN 0-87969-624-9, "Microarray biochip Technology "M Schena, 2000, ISBN 1-881299-37-6).

The Disadvantages of the prior art have been through a new method the preparation of the surface overcome a solid phase.

Short description of Invention:

The application describes a surface of a solid phase which has a very low unspecific binding of labeled components such as labeled nucleotides and labeled nucleic acids ( 1A -D), s. Test 1.1 to 1.4 Examples 1.1.

Nucleic acid chains can be specifically bound to the surface of the solid according to the invention. The surface of the invention with fixed nucleic acids fixed phase can be used in methods for the analysis of nucleic acids, the hybridization ( 1E , Example 5.1.2) of complementary labeled nucleic acids and / or enzymatic reactions on fixed nucleic acid chains ( 1F , Example 6.1), wherein both hybridization of labeled nucleic acids and enzymatic reactions with fixed nucleic acid chains can be repeated several times. In this case, the background signal, which has arisen during the single or repeated contact between the solid phase and the labeled components due to the non-specific binding, remains low. These properties of the surface enable better performance of the processes compared to the results that can be achieved with conventional surfaces.

The solid phase in one embodiment has silica (SiO ₂ ) as the main component. Glass or quartz are preferred forms of silica.

In another embodiment, the solid phase has as its main component pure silicon, wherein the outer layer is oxidized to SiO ₂ .

In an embodiment is the solid phase on a carrier attached. The carrier For example, it is made of plastic.

The surface the solid phase is preferably flat. It is in one embodiment a component of a device suitable for the exchange of solutions is, for example, a flow cell.

In an embodiment is the solid phase or its components for electromagnetic radiation for wavelengths between 400 and 1000 nm transparent. In one embodiment, the transparency for all wavelength in this area, in another embodiment, it is for waves only certain lengths transparent, so that the solid phase has a filter function.

• 1. Entfernung der Außenschicht der festen Phase, wobei eine regenerierte Oberfläche der festen Phase entsteht, die eine SiO₂ Oberfläche ist.
• 2. Kopplung von Nukleinsäureketten an die regenerierte Oberfläche der festen Phase

The preparation of the surface of the solid phase in one embodiment includes the following essential steps:

• 1. Removal of the outer layer of the solid phase, wherein a regenerated surface of the solid phase is formed, which is a SiO ₂ surface.
• 2. Coupling of nucleic acid chains to the regenerated surface of the solid phase

• 1. Entfernung der Außenschicht der festen Phase, wobei eine regenerierte Oberfläche der festen Phase entsteht, die eine SiO₂ Oberfläche ist.
• 2. Kopplung einer Linker-Komponente an die regenerierte Oberfläche der festen Phase,
• 3. Kopplung von Nukleinsäureketten an die Linker-Komponente.

In a further embodiment, the preparation includes the following essential steps:

• 1. Removal of the outer layer of the solid phase, wherein a regenerated surface of the solid phase is formed, which is a SiO ₂ surface.
• 2. Coupling of a linker component to the regenerated surface of the solid phase,
• 3. Coupling of nucleic acid chains to the linker component.

The Removal of the outer layer takes place by chemical reaction in an aqueous or aqueous-organic Solution. In one embodiment becomes the regenerated surface not in the further course of the manufacturing and analysis process dried out. These steps can be integrated into a manufacturing process, which includes more Includes steps for editing the surface.

In an embodiment the manufacturing steps take place only on a part of the surface of the solid phase. The remaining surface of the solid phase is in this embodiment protected from treatment.

In an embodiment have the nucleic acid chains bound to the solid phase an anchor function, in another embodiment of the application the bound nucleic acids a primer function.

The produced according to the invention surfaces the solid phase can for example, in a method for high parallel sequencing used by individual nucleic acid chain molecules for example (WO 02088382, WO 03020968, WO 02072892).

there is used in the preparation for sequencing from the genetic Material first a population of relatively small, single-stranded nucleic acid chain fragments (NSKFs) carrying a primer binding site. Then be these fragments to the fixed on the solid phase nucleic acid chains hybridized with primer function, with extension-capable primer-template complexes arise.

The NSKFs become random when immobilized or bound the surface distributed, i. So it does not have to be exact positioning the individual chains are respected. The NSKF primer complexes have to be included be fixed in such a density on the surface that a unique assignment of the later detected signals from the incorporated labeled nucleotides guaranteed to individual NSKFs is.

• a) Zugabe einer Lösung mit markierten Nukleotiden (NTs*) und Polymerase zu den gebundenen NSKF-Primer-Komplexen,
• b) Inkubation der gebundenen NSKF-Primer-Komplexe mit dieser Lösung unter Bedingungen, die zur enzymatischen Kopplung markierter Nukleotide geeignet sind,
• c) Waschen,
• d) Detektion der Signale von einzelnen eingebauten markierten Nukleotiden,
• e) Entfernung der Markierung von den eingebauten Nukleotiden,
• f) Waschen.

After preparation of the NSKFs, the sequencing reaction is started with all NSKF primer complex molecules immobilized on the surface. The basis of sequencing is the synthesis of the complementary strand to each individual bound NSKF. In this case, labeled nucleotides are incorporated into the newly synthesized strand. The sequencing reaction proceeds in several cycles. For example, a cycle includes the following steps:

• a) Addition of a solution with labeled nucleotides (NTs *) and polymerase to the bound NSKF primer complexes,
• b) incubation of the bound NSKF-primer complexes with this solution under conditions suitable for the enzymatic coupling of labeled nucleotides,
• c) washing,
• d) detection of signals from individual incorporated labeled nucleotides,
• e) removal of the label from the incorporated nucleotides,
• f) washing.

Possibly a multiple repetition of the cycle (a-f) takes place.

After that is for each pinned NSKF has its specific sequence out of order the built-in NTs * determined.

The Number of performed Cycles depends thereby from the respective task off, is theoretically not limited and is preferably between 5 and 500.

The Process is carried out, for example, with an apparatus which in application WO 03031947. The local resolution this apparatus (total resolution, inclusively optical and electronic local Resolution) is preferably between 0.2 and 2 microns.

The Surface of the invention solid phase has a very low non-specific binding of labeled Components. This allows the implementation from many incubation steps with labeled components, for example a repeated longer Contact between the labeled nucleotides and the surface. The Density of signals from nonspecifically bound labeled components stays during the Sequencing method low and interferes only slightly with the specific signals.

Especially is such a surface preferred in the methods containing more than 3 incubation steps with tagged components.

• – Oberfläche einer festen Phase mit einer geringen unspezifischen Bindung von markierten und unmarkierten Nukleotiden und/oder markierten und unmarkierten Nukleinsäuren,
• – Oberfläche einer festen Phase mit einer geringen unspezifischen Bindung von markierten und unmarkierten Nukleotiden und/oder markierten und unmarkierten Nukleinsäuren, wobei an der Oberfläche der festen Phase Nukleinsäureketten spezifisch gebunden sind,
• – Oberfläche einer festen Phase mit einer geringen unspezifischen Bindung von markierten und unmarkierten Nukleotiden und/oder markierten und unmarkierten Nukleinsäuren, wobei an der Oberfläche Nukleinsäureketten spezifisch gebunden sind, die an einer Hybridisierungsreaktion mit komplementären Nukleinsäuresträngen teilnehmen können.
• – Oberfläche einer festen Phase mit einer geringen unspezifischen Bindung von markierten und unmarkierten Nukleotiden und/oder markierten und unmarkierten Nukleinsäuren, die in einem Verfahren zur hochparallelen Analyse von Nukleinsäuresequenzen eingesetzt werden kann,
• – Methoden zur Herstellung solcher Oberflächen einer festen Phase
• – Methoden zur spezifischen Bindung von Nukleinsäureketten an solche Oberflächen einer festen Phase
• – Methode zur Sequenzierung von Nukleinsäurenketten an solchen Oberflächen einer festen Phase
• – Vorrichtung zum Austausch von Flüssigkeiten, die solche Oberflächen einschließt
• – Vorteilhafte Ausführungen für Verfahren zur Sequenzierung von Nukleinsäuren mit der erfindungsgemäßen Oberfläche
• – Vorteilhafte Kombination von markierten Komponenten wie markierte Nukleotide und markierte Nukleinsäuren und der erfindungsgemäßen Reaktionsoberfläche.

The following topics are displayed in the application:

• Surface of a solid phase with a low unspecific binding of labeled and unlabelled nucleotides and / or labeled and unlabeled nucleic acids,
• Surface of a solid phase with a low unspecific binding of labeled and unlabelled nucleotides and / or labeled and unlabeled nucleic acids, nucleic acid chains being specifically bound to the surface of the solid phase,
• - Surface of a solid phase with a low non-specific binding of labeled and unlabelled nucleotides and / or labeled and unlabeled nucleic acids, wherein on the surface nucleic acid chains are specifically bound, which can participate in a hybridization reaction with complementary nucleic acid strands.
• Surface of a solid phase with a low unspecific binding of labeled and unlabelled nucleotides and / or labeled and unlabeled nucleic acids, which can be used in a method for the highly parallel analysis of nucleic acid sequences,
• - Methods for producing such surfaces of a solid phase
• Methods for specific binding of nucleic acid chains to such surfaces of a solid phase
• - Method for sequencing nucleic acid chains on such surfaces of a solid phase
• - Device for the exchange of liquids, which includes such surfaces
• Advantageous embodiments for methods for sequencing nucleic acids with the surface according to the invention
• Advantageous combination of labeled components such as labeled nucleotides and labeled nucleic acids and the reaction surface of the invention.

• 1. Oberfläche einer festen Phase für Verfahren zur Analyse einer Vielzahl von Nukleinsäuremolekülen mit optischen Mitteln.
• 2. Feste Phase für Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln, wobei die Oberfläche der festen Phase eine Eigenschaft der geringen unspezifischen Bindung von markierten Komponenten aufweist.
• 3. Feste Phase nach Aspekt 1 oder 2, wobei die unspezifische Bindung von markierten Komponenten eine Bindung von weniger als 200 Ereignisse/100 μm² während der Analyse beträgt. Als Ereignisse der unspezifischen Bindung werden hier detektierbare Signale von einzelnen markierten Komponenten verstanden. Dabei können markierte Komponenten ein oder mehrere signalgebende Moleküle aufweisen.
• 4. Feste Phase nach Aspekt 1 oder 2, wobei die unspezifische Bindung von markierten Komponenten eine Bindung von weniger als 100 Ereignisse/100 μm² während der Analyse beträgt.
• 5. Feste Phase nach Aspekt 1 oder 2, wobei die unspezifische Bindung von markierten Komponenten eine Bindung von weniger als 50 Ereignisse/100 μm² während der Analyse beträgt.
• 6. Feste Phase nach Aspekt 1 oder 2, wobei die unspezifische Bindung von markierten Komponenten eine Bindung von weniger als 20 Ereignisse/100 μm² während der Analyse beträgt.
• 7. Feste Phase nach Aspekt 1 oder 2, wobei die unspezifische Bindung von markierten Komponenten eine Bindung von weniger als 10 Ereignisse/100 μm² während der Analyse beträgt.
• 8. Feste Phase nach Aspekt 1 oder 2, wobei die unspezifische Bindung von markierten Komponenten eine Bindung von weniger als 5 Ereignisse/100 μm² während der Analyse beträgt.
• 9. Feste Phase nach Aspekt 1 oder 2, wobei die unspezifische Bindung von markierten Komponenten eine Bindung von weniger als 2 Ereignisse/100 μm² während der Analyse beträgt.
• 10. Feste Phase nach Aspekten 1 bis 9, wobei die feste Phase eine Silica-Oberfläche einschließt.
• 11. Feste Phase nach Aspekten 1 bis 9, wobei die feste Phase eine Glas- oder Quarz-Oberfläche einschließt.
• 12. Feste Phase nach Aspekten 1 bis 9, wobei die feste Phase eine SiO₂-Oberfläche einschließt. Die feste Phase kann in einer Ausführungsform einen Anteil von SiO₂ aufweisen, der in folgenden Bereichen liegt: zwischen 1% und 10%, zwischen 10% und 50%, zwischen 50% und 80%, zwischen 80% und 100%.
• 13. Feste Phase nach Aspekten 1 bis 9, wobei die feste Phase eine Si-OH-Oberfläche einschließt.
• 14. feste Phase nach Aspekten 1 bis 13, wobei die Oberfläche der festen Phase flach ist.
• 15. Feste Phase nach Aspekten 1 bis 14, wobei an der Oberfläche dieser festen Phase Nukleinsäureketten fixiert sind.
• 16. Feste Phase nach Aspekten 1 bis 14, wobei an diese feste Phase Nukleinsäureketten über eine Linker-Komponente fixiert sind.
• 17. Methode zur Herstellung der festen Phase nach Aspekten 1 bis 16, wobei die Herstellung folgende Schritte einschließt; 1. Bereitstellung einer regenerierten Oberfläche einer festen Phase durch die Entfernung der Außenschicht der festen Phase 2. Kopplung von Nukleinsäureketten an die regenerierte Oberfläche der festen Phase
• 18. Methode zur Herstellung der festen Phase nach Aspekten 1 bis 16, wobei die Herstellung folgende Schritte einschließt; 1. Bereitstellung einer regenerierten Oberfläche einer festen Phase durch die Entfernung der Außenschicht der festen Phase 2. Kopplung einer Linker-Komponente an die regenerierte Oberfläche der festen Phase 3. Kopplung von Nukleinsäureketten an die Linker-Komponente
• 19. Methode zur Herstellung der festen Phase nach Aspekten 1 bis 16, wobei die Herstellung folgende Schritte einschließt: 1. Bereitstellung einer regenerierten Oberfläche einer festen Phase durch die Entfernung der Außenschicht der festen Phase 2. chemische Synthese von Nukleinsäureketten an der regenerierten Oberfläche der festen Phase
• 20. Methode zur Herstellung der festen Phase nach Aspekten 1 bis 16, wobei die Herstellung folgende Schritte einschließt: 1. Bereitstellung einer regenerierten Oberfläche einer festen Phase durch die Entfernung der Außenschicht der festen Phase 2. Kopplung einer Linker-Komponente an die regenerierte Oberfläche der festen Phase 3. chemische Synthese von Nukleinsäureketten an der festen Phase. Viele Verfahren zur Festphasensynthese von Nukleinsäureketten sind bekannt, z.B. mit konventioneller Oligonukleotidsythese (Capaldi et al., 2000; Damha et al., 1990; Devivar et al., 1999; Habus et al., 1998; Pon, 1993; Pon et al., 1988; Sinha, 1993; Song et al., 2003; Veiko et al., 1991; Vu et al., 1990)
• 21. Methode zur Herstellung der festen Phase nach Aspekten 1 bis 16, wobei die Herstellung folgende Schritte einschließt: 1. Bereitstellung einer regenerierten Oberfläche einer festen Phase durch die Entfernung der Außenschicht der festen Phase 2. Kopplung von Nukleinsäureketten an die regenerierte Oberfläche der festen Phase 3. Kopplung weiterer Substanzen an die regenerierte Oberfläche der festen Phase
• 22. Methode zur Herstellung der festen Phase nach Aspekten 1 bis 16, wobei die Herstellung folgende Schritte einschließt: 1. Bereitstellung einer regenerierten Oberfläche einer festen Phase durch die Entfernung der Außenschicht der festen Phase 2. Kopplung einer Linker-Komponente an die regenerierte Oberfläche der festen Phase 3. Kopplung von Nukleinsäureketten an die Linker-Komponente 4. Kopplung weiterer Substanzen an die regenerierte Oberfläche oder an die Linker-Komponente
• 23. Methode zur Herstellung der festen Phase nach Aspekten 1 bis 16, wobei die Herstellung folgende Schritte einschließt: 1. Bereitstellung einer regenerierten Oberfläche einer festen Phase durch die Entfernung der Außenschicht der festen Phase 2. chemische Synthese von Nukleinsäureketten an der regenerierten Oberfläche der festen Phase 3. Kopplung weiterer Substanzen an die regenerierte Oberfläche der festen Phase
• 24. Methode zur Herstellung der festen Phase nach Aspekten 1 bis 16, wobei die Herstellung folgende Schritte einschließt: 1. Bereitstellung einer regenerierten Oberfläche einer festen Phase durch die Entfernung der Außenschicht der festen Phase 2. Kopplung einer Linker-Komponente an die regenerierte Oberfläche der festen Phase 3. chemische Synthese von Nukleinsäureketten an der regenerierten Oberfläche der festen Phase 4. Kopplung weiterer Substanzen an die regenerierte Oberfläche oder an die Linker-Komponente
• 25. Methode zur Herstellung der festen Phase nach Aspekten 1 bis 24, wobei die Außenschicht nur an einem Teil der festen Phase abgetragen wird. In einer Ausführungsform kann die Entfernung auch nur an einem Teil der Oberfläche der festen Phase vorgenommen werden. Ebenso kann die Kopplung der Nukleinsäureketten an einem Anteil der bereitgestellten Oberfläche der festen Phase vorgenommen werden.
• 26. Methode zur Herstellung der festen Phase nach Aspekten 1 bis 25, wobei die Entfernung der Außenschicht durch chemische Reaktion erfolgt
• 27. Methode zur Herstellung der festen Phase nach Aspekt 26, wobei die chemische Reaktion in Anwesenheit von HF erfolgt. Dem Fachmann sind viele chemische Kombinationen (Lösungen) bekannt, die HF enthalten und zur Abtragung der äußeren SiO₂-Schicht dienen. Beispielsweise sind gepufferte HF-Lösungen (z.B. HF und NH₄F) oder auch Mischungen aus HF, HNO₃ und CH₃COOH zur Entfernung der äußeren Schicht geeignet. Die Lösungen können dabei wässrig oder wässrig-organisch sein.
• 28. Methode zur Herstellung der festen Phase nach Aspekt 26, wobei die chemische Reaktion in Anwesenheit von Hydroxiden erfolgt. Dem Fachmann sind viele Hydroxide bekannt, die zur Entfernung der äußeren SiO₂-Schicht dienen können. Beispielsweise können Lösungen verwendet werden, die NaOH, KOH, NH₄OH, LiOH enthalten. Die Lösungen können dabei wässrig oder wässrig-organisch sein.
• 29. Methode zur Herstellung der festen Phase nach Aspekten 1 bis 28, wobei die Dicke der entfernten Außenschicht zwischen 1 nm und 10 nm liegt.
• 30. Methode zur Herstellung der festen Phase nach Aspekten 1 bis 28, wobei die Dicke der entfernten Außenschicht zwischen 10 nm und 100 nm liegt.
• 31. Methode zur Herstellung der festen Phase nach Aspekten 1 bis 28, wobei die Dicke der entfernten Außenschicht zwischen 100 nm und 1 μm liegt.
• 32. Methode zur Herstellung der festen Phase nach Aspekten 1 bis 28, wobei die Dicke der entfernten Außenschicht zwischen 1 μm und 10μm liegt.
• 33. Methode zur Herstellung der festen Phase nach Aspekten 1 bis 28, wobei die Dicke der entfernten Außenschicht zwischen 10 μm und 100 μm liegt.
• 34. Methode zur Herstellung der festen Phase nach Aspekten 1 bis 28, wobei nach der Entfernung der Außenschicht der festen Phase keine Trocknungsschritte erfolgen und weitere Schritte in der flüssigen Phase stattfinden. Unter weiteren Schritten werden hier sowohl Herstellungsschritte, wie beispielsweise spezifische Kopplung von Linker-Komponenten oder Fixierung von Nukleinsäureketten an die regenerierte Oberfläche der festen Phase, als auch Schritte während der Analyse wie z.B. Inkubationsschritte, Waschschritte usw. Die flüssige Phase kann dabei beispielsweise wässrige Lösungen oder wässrig-organische Lösungen oder auch organische Lösungen einschließen. Die Lösungen können Salze enthalten und können Puffereigenschaften besitzen.
• 35. Methode zur Herstellung der festen Phase nach Aspekten 21 bis 24, wobei die Gruppe der "weiteren Substanzen" folgende Substanzen einschließen: Monomere (insbesondere Substanzen, die eine oder mehrere Phosphat-, Sulfat-, Carboxy-Gruppen tragen), Polymere (Polyethylenglycol, Polyacrylate, Polyacrylamide, Polyphosphate, Proteine)
• 36. Feste Phase für ein Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln, die nach Methoden in Aspekten 17 bis 35 hergestellt wird.
• 37. Feste Phase für ein Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln, die nach Methoden in Aspekten 17 bis 35 hergestellt wird, wobei an der Oberfläche Nukleinsäureketten fixiert sind.
• 38. Feste Phase nach Aspekten 1 bis 37 mit fixierten Nukleinsäureketten, wobei fixierte Nukleinsäureketten eine einheitliche Population darstellen Die an der Oberfläche fixierte Nukleinsäureketten können eine Anker- oder eine Primerfunktion haben. In einer Ausführungsform können die zu analysierenden Nukleinsäureketten an die auf der regenerierten Oberfläche der festen Phase fixierten Nukleinsäureketten hybridisiert werden. In einer weiteren Ausführungsform kann an den gebildeten Primer-Matrizenkomplexen eine enzymatische Reaktion durchgeführt werden.
• 39. Feste Phase nach Aspekten 1 bis 37 mit fixierten Nukleinsäureketten, wobei fixierte Nukleinsäureketten mehrere unterschiedliche Populationen darstellen In einer Ausführungsform werden unterschiedlichen Populationen auf der festen Phase in einer vorgegebenen Anordnung fixiert, wobei ein Array entsteht. In einer anderen Ausführungsform werden unterschiedliche Populationen in einer zufälligen Anordnung an der Oberfläche fixiert (WO 02088382, WO 02072892). Die an der Oberfläche fixierte Nukleinsäureketten können eine Anker- oder eine Primerfunktion haben. In einer Ausführungsform können die zu analysierenden Nukleinsäureketten an die auf der regenerierten Oberfläche der festen Phase fixierten Nukleinsäureketten hybridisiert werden. In einer weiteren Ausführungsform kann an den gebildeten Primer-Matrizenkomplexen eine enzymatische Reaktion durchgeführt werden.
• 40. Feste Phase nach Aspekten 1 bis 39 mit fixierten Nukleinsäureketten, wobei Nukleinsäureketten eine Länge zwischen 5 und 50 Nukleotiden haben
• 41. Feste Phase nach Aspekten 1 bis 39 mit fixierten Nukleinsäureketten, wobei Nukleinsäureketten eine Länge zwischen 20 und 200 Nukleotiden haben
• 42. Feste Phase nach Aspekten 1 bis 39 mit fixierten Nukleinsäureketten, wobei Nukleinsäureketten eine Länge zwischen 50 und 500 Nukleotiden haben
• 43. Feste Phase mit fixierten Nukleinsäureketten nach Aspekten 1 bis 42, wobei die Fixierung von Nukleinsäureketten kovalent ist.
• 44. Feste Phase mit fixierten Nukleinsäureketten nach Aspekten 1 bis 42, wobei die Fixierung affin ist.
• 45. Feste Phase nach Aspekten 16, 18, 20, 22, 24, wobei der Linker eine Länge von 1 bis 50 nm hat
• 46. Feste Phase nach Aspekten 16, 18, 20, 22, 24, 45, wobei der Linker ein nicht verzweigtes Polymer ist
• 47. Feste Phase nach Aspekten 16, 18, 20, 22, 24, 45 wobei der Linker ein verzweigtes Polymer ist
• 48. Feste Phase nach Aspekten 16, 18, 20, 22, 24, wobei Streptavidin oder Avidin als Linker auftritt.
• 49. Feste Phase nach Aspekten 16, 18, 20, 22, 24, wobei Nukleinsäureketten als Linker auftreten.
• 50. Feste Phase mit fixierten Nukleinsäureketten nach Aspekten 1 bis 49, wobei die Dichte der fixierten Nukleinsäureketten zwischen 1/100 μm² und 10/100 μm² liegt.
• 51. Feste Phase mit fixierten Nukleinsäureketten nach Aspekten 1 bis 49, wobei die Dichte der fixierten Nukleinsäureketten zwischen 10/100 μm² und 100/100 μm² liegt.
• 52. Feste Phase mit fixierten Nukleinsäureketten nach Aspekten 1 bis 49, wobei die Dichte der fixierten Nukleinsäureketten zwischen 100/100 μm² und 1000/100 μm² liegt.
• 53. Feste Phase mit fixierten Nukleinsäureketten nach Aspekten 1 bis 49, wobei die Dichte der fixierten Nukleinsäureketten zwischen 1000/100 μm² und 10.000/100 μm² liegt.
• 54. Feste Phase mit fixierten Nukleinsäureketten nach Aspekten 1 bis 49, wobei die Dichte der fixierten Nukleinsäureketten zwischen 10.000/ 100 μm² und 1.000.000/100 μm² liegt. Die an der Oberfläche fixierte Nukleinsäureketten können eine Anker- oder eine Primerfunktion haben. In einer Ausführungsform können die zu analysierenden Nukleinsäureketten an die auf der regenerierten Oberfläche der festen Phase fixierten Nukleinsäureketten komplementär hybridisiert werden. In einer weiteren Ausführungsform kann an den gebildeten Primer-Matrizenkomplexen eine enzymatische Reaktion durchgeführt werden. Die Dichte der extensionsfähigen Primer-Matrizenkomplexe liegt vorzugsweise in einem der folgenden Bereiche: 1/100 μm² bis 10 / 100 μm², 10/100 μm² bis 20/100 μm², 20/100 μm² bis 30/100 μm², 30/100 μm² bis 40/100 μm², 40/100 μm² bis 50/100 μm², 50/100 μm² bis 60/100 μm², 60/100 μm² bis 70/100 μm², 70/100 μm² bis 80/100 μm², 80/100 μm² bis 90/100 μm², 90/100 μm² bis 100/100 μm², 100/100 μm² bis 120/100 μm², 120/100 μm² bis 130/100 μm², 130/100 μm² bis 140/100 μm², 140/100 μm² bis 150/100 μm², 150/100 μm² bis 160/100 μm², 160/100 μm² bis 170/100 μm², 170/100 μm² bis 180/100 μm², 180/100 μm² bis 190/100 μm², 190/100 μm² bis 200/100 μm², 200/100 μm² bis 250/100 μm², 250/100 μm² bis 300/100 μm², 300/100 μm² bis 400/100 μm². In einer Ausführungsform ist die Dichte der an der regenerierten Oberfläche der festen Phase fixierten Nukleinsäureketten über die gesamte Oberfläche der festen Phase gleich. In einer anderen Ausführungsform schließt die hergestellte feste Phase Bereiche ein, die unterschiedliche Dichte der fixierten Nukleinsäureketten aufweisen. Dies ist insbesondere dann vorteilhaft, wenn unbekannte Konzentrationen von zu analysierenden Nukleinsäureketten in einem Verfahren wie (WO 02088382, WO 02072892) eingesetzt werden.
• 55. Feste Phase mit fixierten Nukleinsäureketten nach Aspekten 1 bis 54, wobei an der festen Phase ein erkennbares Muster angebracht ist. Dieses Muster kann beispielsweise eine Markierung sein, die mit optischen Mitteln erkannt werden kann.
• 56. Feste Phase mit fixierten Nukleinsäureketten nach Aspekten 1 bis 55, wobei die feste Phase ein Teil einer Vorrichtung zum Austausch von Flüssigkeiten darstellt.
• 57. Feste Phase mit fixierten Nukleinsäureketten nach Aspekten 1 bis 56, wobei die feste Phase für elektromagnetische Strahlung mit Wellenlängen im Bereich zwischen 200 nm bis 400 nm durchlässig ist.
• 58. Feste Phase mit fixierten Nukleinsäureketten nach Aspekten 1 bis 56, wobei die feste Phase für elektromagnetische Strahlung mit Wellenlängen im Bereich zwischen 200 nm bis 2000 nm durchlässig ist.
• 59. Feste Phase mit fixierten Nukleinsäureketten nach Aspekten 1 bis 56, wobei die feste Phase für elektromagnetische Strahlung mit Wellenlängen im Bereich zwischen 400 nm bis 800 nm durchlässig ist. Feste Phase kann bestimmte Bereiche der elektromagnetischen Strahlung absorbieren und somit eine Filterfunktion besitzen.
• 60. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln, wobei die feste Phase nach Aspekten 1 bis 59 verwendet wird.
• 61. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekt 60, wobei markierte Komponenten verwendet werden.
• 62. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekt 61, wobei die Markierung von den markierten Komponenten durch Abspaltung entfernt werden kann. Die Abspaltung kann dabei chemisch, photochemisch oder auch enzymatisch erfolgen. Die Abspaltung kann beispielsweise in einer wässrigen oder in einer wässrig-organischen Phase erfolgen.
• 63. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 61 und 62, wobei markierte Komponenten markierte Ribonukleosidtriphosphate oder Deoxyribonucleosidtriphosphate oder Dideoxyribonukleosidtriphosphate einschließen. Dabei können die sowohl in der Natur vorkommenden Nukleoside, wie Adenosin, Guanosin, Thymidin, Uridin, Cytosin als auch deren Modifikationen, Nukleotidanaloga, wie z.B. 7-Deazaadenosin oder 7-Deazaguanosin verwendet werden. Die Markierung kann am Nukleotid an der Base, am Zucker oder an dem Phosphat-Anteil gekoppelt sein.
• 64. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 61 bis 63, wobei markierte Komponenten reversibel markierte Ribonukleosidtriphosphate oder Deoxyribonucleosidtriphosphate oder Dideoxyribonukleosidtriphosphate einschließen.
• 65. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 61 und 62, wobei markierte Komponenten markierte Nukleinsäuren einschließen.
• 66. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekt 65, wobei markierte Komponenten reversibel markierte Nukleinsäuren einschließen.
• 67. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 66, wobei parallel 100 bis 10.000 Nukleinsäureketten analysiert werden
• 68. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 66, wobei parallel 1000 bis 100.000 Nukleinsäureketten analysiert werden
• 69. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 66, wobei parallel 10.000 bis 1.000.000 Nukleinsäureketten analysiert werden
• 70. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 66, wobei parallel 100.000 bis 10.000.000 Nukleinsäureketten analysiert werden
• 71. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 66, wobei parallel 1.000.000 bis 100.000.000 Nukleinsäureketten analysiert werden
• 72. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 66, wobei parallel 100.000.000 bis 10.000.000.000 Nukleinsäureketten analysiert werden
• 73. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 72, wobei Ereignisse an einzelnen Nukleinsäurekettenmolekülen optisch detektiert werden
• 74. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 73, wobei optische Mittel eine Auflösung bis zu 0,3 μm ermöglichen
• 75. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 73, wobei optische Mittel eine Auflösung bis zu 0,5 μm ermöglichen
• 76. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 73, wobei optische Mittel eine Auflösung bis zu 0,8 μm ermöglichen
• 77. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 73, wobei optische Mittel eine Auflösung bis zu 1 μm ermöglichen
• 78. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 73, wobei optische Mittel eine Auflösung bis zu 1,2 μm ermöglichen
• 79. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 73, wobei optische Mittel eine Auflösung bis zu 1,5 μm ermöglichen
• 80. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 73, wobei optische Mittel eine Auflösung bis zu 2 μm ermöglichen
• 81. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 80, wobei die Analyse durch die Synthese eines komplementären Stranges verläuft (zyklische Sequenzierung).
• 82. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekt 81, wobei die Analyse durch die zyklische Sequenzierung verläuft und folgende Schritte einschließt: 1. Bereitstellung einer festen Phase nach Aspekten 1 bis 59 2. Bindung von zu analysierenden Nukleinsäuremolekülen an diese feste Phase unter Ausbildung extensionsfähiger Primer-Matrize-Komplexen, 3. Durchführung einer zyklischen Reaktion mit den auf der festen Phase fixierten Nukleinsäureketten, die folgende Schritte einschließt: 3.1 Inkubation einer Lösung mit einer oder mehreren Polymerase und einem oder mehreren markierten Nukleotiden (Polymerase-Nukleotid-Gemisch) mit der festen Phase unter Bedingungen, die eine Einbaureaktion von Nukleotiden an den gebildeten Primer-Matrize-Komplexen zulassen, 3.2 Entfernung des Polymerase-Nukleotid-Gemischs und Waschen der festen Phase 3.3 Detektion der Signale von eingebauten markierten Nukleotiden, wobei relative Koordinaten einzelne Signale identifiziert werden 3.4 Entfernung der Signale von eingebauten Nukleotiden, 3.5 gegebenenfalls Waschen der festen Phase 3.6 gegebenenfalls Wiederholung der Schritte 3.1 bis 3.5 4. Rekonstruktion der Sequenzen von einzelnen Nukleinsäuremolekülen aus den unter Schritt 3.3 erhaltenen Signalen
• 83. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 82, wobei die zu analysierenden Nukleinsäuremoleküle Nukleinsäureketten mit einer Länge zwischen 30 und 300 Nukleotide darstellen.
• 84. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 82, wobei die zu analysierenden Nukleinsäuremoleküle Nukleinsäureketten mit einer Länge zwischen 30 und 3000 Nukleotide darstellen.
• 85. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 84, wobei die zu analysierenden Nukleinsäuremoleküle Deoxyribonukleinsäureketten oder Ribonukleinsäureketten darstellen.
• 86. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 85, wobei die bereitgestellte feste Phase fixierte Nukleinsäuren trägt, die als Primer für die Sequenzierung dienen.
• 87. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekt 86, wobei die bereitgestellte feste Phase fixierte Nukleinsäuren trägt, die als Primer für die Sequenzierung dienen und die zu analysierenden Nukleinsäuremoleküle direkt an diese Primer hybridisiert werden, wobei extensionsfähige Primer-Matrize-Komplexe gebildet werden.
• 88. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 85, wobei zunächst die zu analysierenden Nukleinsäuremoleküle an die feste Phase fixiert werden und anschließend ein Primer an die zu analysierenden Nukleinsäureketten hybridisiert wird.
• 89. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 82, wobei die Fixierung von zu analysierenden Nukleinsäuremolekülen kovalent an die feste Phase erfolgt.
• 90. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 82, wobei die Fixierung von zu analysierenden Nukleinsäuremolekülen affin an die feste Phase erfolgt.
• 91. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 82 und 90, wobei die bereitgestellte feste Phase fixierte Nukleinsäureketten trägt, die als Anker für affine Fixierung von zu analysierenden Nukleinsäuremolekülen dienen.
• 92. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 82 und 90, wobei die Fixierung von zu analysierenden Nukleinsäureketten durch die Hybridisierung bzw. affine Bindung an den Anker an der festen Phase erfolgt.
• 93. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 92, wobei die Dichte der fixierten extensionsfähigen Primer-Matrize-Komplexe auf der festen Phase zwischen 5 und 50 Komplexe pro 100 μm² liegt.
• 94. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 92, wobei die Dichte der fixierten extensionsfähigen Primer-Matrize-Komplexe auf der festen Phase zwischen 50 und 250 Komplexe pro 100 μm² liegt.
• 95. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 94, wobei die zu analysierenden Nukleinsäuremoleküle an die feste Phase in zufälliger Anordnung fixiert werden
• 96. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 60 bis 94, wobei die zu analysierenden Nukleinsäuremoleküle an die feste Phase in vorbestimmter Anordnung fixiert werden
• 97. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 82 bis 96, wobei im Schritt 3.1 verwendete Nukleotide markierte Ribonukleosidtriphosphate oder Deoxyribonucleosidtriphosphate oder Dideoxyribonukleosidtriphosphate einschließen.
• 98. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 82 bis 97, wobei im Schritt 3.1 verwendete Nukleotide eine reversibel terminierende Gruppe tragen, so dass nur ein markiertes Nukleotid in einem Inkubationsschritt 3.1 eingebaut werden kann.
• 99. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekt 98, wobei nach dem Detektionsschritt 3.3 ein weiterer Schritt zur Entfernung der reversibel terminierenden Gruppe im durchgeführt wird, so dass die Extensionsreaktion im weiteren Verlauf stattfinden kann.
• 100. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 82 bis 99, wobei verwendete Nukleotide eine einheitliche Markiertung tragen und im Inkubationsschritt 3.1 modifizierte Nukleotide gleicher Basenart zugegeben werden.
• 101. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 82 bis 99, wobei verwendete Nukleotide eine basenspezifische Markiertung tragen und im Inkubationsschritt 3.1 modifizierte Nukleotide unterschiedlicher Basenart zugegeben werden.
• 102. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 82 bis 101, wobei im Schritt 3.3 relative Intensität eines jeden Signals bestimmt wird.
• 103. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 82 bis 101, wobei die Zahl der Wiederholungen der Schritte 3.1 bis 3.5 zwischen 2 und 10 liegt.
• 104. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 82 bis 101, wobei die Zahl der Wiederholungen der Schritte 3.1 bis 3.5 zwischen 10 und 25 liegt.
• 105. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 82 bis 101, wobei die Zahl der Wiederholungen der Schritte 3.1 bis 3.5 zwischen 25 und 50 liegt.
• 105. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekten 82 bis 101, wobei die Zahl der Wiederholungen der Schritte 3.1 bis 3.5 zwischen 50 und 200 liegt.
• 106. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekt 60 bis 80, wobei die Analyse durch Hybridisierung komplementärer markierter Nukleinsäureketten verläuft.
• 107. Verfahren zur Analyse von Nukleinsäureketten mit der festen Phase nach Aspekten 1 bis 59, wobei die zu analysierenden Nukleinsäureketten an der festen Phase fixiert sind und die Analyse durch die Hybridisierung von komplementären markierten Nukleinsäureketten erfolgt.
• 108. Verfahren zur Analyse von Nukleinsäureketten mit der festen Phase nach Aspekten 1 bis 59, wobei an der festen Phase Nukleinsäurestränge in einer vordefinierten Anordnung fixiert sind und die zu analysierenden Nukleinsäureketten durch die Hybridisierung an die fixierten Nukleinsäurestränge analysiert werden.

The subject matter of this invention relates to the following aspects:

• 1. A solid phase surface for methods of analyzing a plurality of nucleic acid molecules by optical means.
• 2. A solid phase for methods of parallel analysis of a plurality of individual nucleic acid molecules with optical means, wherein the surface of the solid phase has a property of low non-specific binding of labeled components.
• 3. The solid phase according to aspect 1 or 2, wherein the non-specific binding of labeled components is a binding of less than 200 events / 100 μm ² during the analysis. Non-specific binding events are here understood to be detectable signals from individual labeled components. In this case, labeled components can have one or more signaling molecules.
• 4. The solid phase according to aspect 1 or 2, wherein the non-specific binding of labeled components is a binding of less than 100 events / 100 μm ² during the analysis.
• 5. The solid phase according to aspect 1 or 2, wherein the non-specific binding of labeled components is a binding of less than 50 events / 100 μm ² during the analysis.
• 6. The solid phase according to aspect 1 or 2, wherein the non-specific binding of labeled components is a binding of less than 20 events / 100 μm ² during the analysis.
• 7. Solid phase according to aspect 1 or 2, wherein the non-specific binding of labeled components is a binding of less than 10 events / 100 μm ² during the analysis.
• 8. Solid phase according to aspect 1 or 2, wherein the non-specific binding of labeled components is a binding of less than 5 events / 100 μm ² during the analysis.
• 9. Solid phase according to aspect 1 or 2, wherein the non-specific binding of labeled components is a binding of less than 2 events / 100 μm ² during the analysis.
• 10. Solid phase according to aspects 1 to 9, wherein the solid phase includes a silica surface.
• 11. Solid phase according to aspects 1 to 9, wherein the solid phase includes a glass or quartz surface.
• 12. The solid phase of aspects 1 to 9, wherein the solid phase includes an SiO ₂ surface. In one embodiment, the solid phase may have a content of SiO ₂ in the following ranges: between 1% and 10%, between 10% and 50%, between 50% and 80%, between 80% and 100%.
• 13. Solid phase according to aspects 1 to 9, wherein the solid phase includes a Si-OH surface.
• 14. Solid phase according to aspects 1 to 13, wherein the surface of the solid phase is flat.
• 15. Solid phase according to aspects 1 to 14, wherein on the surface of this solid phase nucleic acid chains are fixed.
• 16. Solid phase according to aspects 1 to 14, wherein nucleic acid chains are attached to this solid phase via a linker component.
• 17. A method of preparing the solid phase according to Aspects 1 to 16, wherein the preparation includes the following steps; 1. Providing a regenerated surface of a solid phase by removing the outer layer of the solid phase 2. Coupling of nucleic acid chains to the regenerated surface of the solid phase
• A method for producing the solid phase according to Aspects 1 to 16, wherein the preparation includes the following steps; 1. Providing a regenerated surface of a solid phase by removing the outer layer of the solid phase. 2. Coupling a linker component to the regenerated surface of the solid phase. 3. Coupling of nucleic acid chains to the linker component
• 19. A method for producing the solid phase according to aspects 1 to 16, wherein the preparation of the following Steps include: 1. Providing a regenerated surface of a solid phase by removing the outer layer of solid phase 2. Chemical synthesis of nucleic acid chains on the regenerated surface of the solid phase
• A method of preparing the solid phase of aspects 1-16, wherein the preparation includes the steps of: 1. providing a regenerated surface of a solid phase by removing the outer layer of the solid phase 2. coupling a linker component to the regenerated surface of the solid phase solid phase 3. chemical synthesis of nucleic acid chains on the solid phase. Many methods for solid phase synthesis of nucleic acid chains are known, for example, with conventional oligonucleotide synthesis (Capaldi et al., 2000, Damha et al., 1990; Devivar et al., 1999; Habus et al., 1998; Pon, 1993; Pon et al. , Sinha, 1993; Song et al., 2003; Veiko et al., 1991; Vu et al., 1990).
• 21. A method of preparing the solid phase of aspects 1-16, wherein the preparation includes the steps of: 1. providing a regenerated surface of a solid phase by removing the outer layer of the solid phase. 2. coupling nucleic acid chains to the regenerated surface of the solid phase 3. Coupling of further substances to the regenerated surface of the solid phase
• A method of preparing the solid phase of aspects 1-16, wherein the preparation includes the steps of: 1. providing a regenerated surface of a solid phase by removing the outer layer of the solid phase. 2. coupling a linker component to the regenerated surface of the solid phase solid phase 3. Coupling of nucleic acid chains to the linker component 4. Coupling of further substances to the regenerated surface or to the linker component
• 23. A method of preparing the solid phase of aspects 1-16, the preparation including the steps of: 1. providing a regenerated surface of a solid phase by removing the outer layer of solid phase 2. chemical synthesis of nucleic acid chains on the regenerated surface of the solid Phase 3. Coupling of further substances to the regenerated surface of the solid phase
• 24. A method of preparing the solid phase of aspects 1-16, wherein the preparation includes the steps of: 1. providing a regenerated surface of a solid phase by removing the outer layer of the solid phase. 2. coupling a linker component to the regenerated surface of the solid phase solid phase 3. Chemical synthesis of nucleic acid chains on the regenerated surface of the solid phase 4. Coupling of further substances to the regenerated surface or to the linker component
• 25. A method for producing the solid phase according to aspects 1 to 24, wherein the outer layer is removed only on a part of the solid phase. In one embodiment, the removal may also be made only on a part of the surface of the solid phase. Likewise, the coupling of the nucleic acid chains can be made on a portion of the provided surface of the solid phase.
• 26. A method for producing the solid phase according to aspects 1 to 25, wherein the removal of the outer layer takes place by chemical reaction
• 27. A method for preparing the solid phase according to aspect 26, wherein the chemical reaction takes place in the presence of HF. The skilled worker is aware of many chemical combinations (solutions) which contain HF and serve to remove the outer SiO ₂ layer. For example, buffered HF solutions (eg HF and NH ₄ F) or else mixtures of HF, HNO ₃ and CH ₃ COOH are suitable for removing the outer layer. The solutions may be aqueous or aqueous-organic.
• 28. A method for preparing the solid phase according to aspect 26, wherein the chemical reaction takes place in the presence of hydroxides. Many hydroxides are known to those skilled in the art, which can serve to remove the outer SiO ₂ layer. For example, solutions containing NaOH, KOH, NH ₄ OH, LiOH can be used. The solutions may be aqueous or aqueous-organic.
• 29. Method for producing the solid phase according to aspects 1 to 28, wherein the thickness of the removed outer layer is between 1 nm and 10 nm.
• 30. Method for producing the solid phase according to aspects 1 to 28, wherein the thickness of the removed Au Outer layer between 10 nm and 100 nm.
• 31. Method for producing the solid phase according to aspects 1 to 28, wherein the thickness of the removed outer layer is between 100 nm and 1 μm.
• 32. Method for producing the solid phase according to aspects 1 to 28, wherein the thickness of the removed outer layer is between 1 μm and 10 μm.
• 33. Method for producing the solid phase according to aspects 1 to 28, wherein the thickness of the removed outer layer is between 10 μm and 100 μm.
• 34. Method for producing the solid phase according to aspects 1 to 28, wherein no drying steps take place after the removal of the outer layer of the solid phase and further steps take place in the liquid phase. Among other steps here are both manufacturing steps, such as specific coupling of linker components or fixation of nucleic acid chains to the regenerated surface of the solid phase, as well as steps during the analysis such as incubation steps, washing steps, etc. The liquid phase can be, for example, aqueous solutions or aqueous-organic solutions or organic solutions include. The solutions may contain salts and may have buffering properties.
• 35. Method for producing the solid phase according to aspects 21 to 24, wherein the group of "further substances" include the following substances: monomers (in particular substances which carry one or more phosphate, sulfate, carboxy groups), polymers (polyethylene glycol , Polyacrylates, polyacrylamides, polyphosphates, proteins)
• 36. A solid phase for a method of parallel analysis of a plurality of individual nucleic acid molecules by optical means, prepared by methods in aspects 17-35.
• 37. A solid phase for a method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means, which is prepared by methods in aspects 17 to 35, wherein on the surface nucleic acid chains are fixed.
• 38. Solid phase according to aspects 1 to 37 with fixed nucleic acid chains, wherein fixed nucleic acid chains constitute a uniform population The nucleic acid chains fixed on the surface can have an anchor or a primer function. In one embodiment, the nucleic acid chains to be analyzed may be hybridized to the nucleic acid chains fixed on the regenerated surface of the solid phase. In a further embodiment, an enzymatic reaction can be carried out on the primer-template complexes formed.
• 39. Solid phase according to aspects 1 to 37 with fixed nucleic acid chains, wherein fixed nucleic acid chains represent several different populations. In one embodiment, different populations are fixed on the solid phase in a given arrangement to form an array. In another embodiment, different populations are fixed in a random arrangement on the surface (WO 02088382, WO 02072892). The nucleic acid chains fixed on the surface may have an anchor or a primer function. In one embodiment, the nucleic acid chains to be analyzed may be hybridized to the nucleic acid chains fixed on the regenerated surface of the solid phase. In a further embodiment, an enzymatic reaction can be carried out on the primer-template complexes formed.
• 40. Solid phase according to aspects 1 to 39 with fixed nucleic acid chains, wherein nucleic acid chains have a length between 5 and 50 nucleotides
• 41. Solid phase according to aspects 1 to 39 with fixed nucleic acid chains, wherein nucleic acid chains have a length between 20 and 200 nucleotides
• 42. Solid phase according to aspects 1 to 39 with fixed nucleic acid chains, wherein nucleic acid chains have a length between 50 and 500 nucleotides
• 43. Fixed phase with fixed nucleic acid chains according to aspects 1 to 42, wherein the fixation of nucleic acid chains is covalent.
• 44. Fixed phase with fixed nucleic acid chains according to aspects 1 to 42, wherein the fixation is affine.
• 45. The solid phase of aspects 16, 18, 20, 22, 24, wherein the linker has a length of 1 to 50 nm
• 46. The solid phase of aspects 16, 18, 20, 22, 24, 45, wherein the linker is a non-branched polymer
• 47. Solid Phase of Aspects 16, 18, 20, 22, 24, 45 wherein the linker is a branched polymer
• 48. Solid phase according to aspects 16, 18, 20, 22, 24, wherein streptavidin or avidin acts as a linker.
• 49. Solid phase according to aspects 16, 18, 20, 22, 24, nucleic acid chains occurring as linker.
• 50. Solid phase with fixed nucleic acid chains according to aspects 1 to 49, wherein the density of the fixed nucleic acid chains is between 1/100 μm ² and 10/100 μm ² .
• 51. Solid phase with fixed nucleic acid chains according to aspects 1 to 49, wherein the density of the fixed nucleic acid chains is between 10/100 μm ² and 100/100 μm ² .
• 52. Solid phase with fixed nucleic acid chains according to aspects 1 to 49, wherein the density of the fixed nucleic acid chains is between 100/100 μm ² and 1000/100 μm ² .
• 53. Solid phase with fixed nucleic acid chains according to aspects 1 to 49, wherein the density of the fixed nucleic acid chains is between 1000/100 μm ² and 10,000 / 100 μm ² .
• 54. Solid phase with fixed nucleic acid chains according to aspects 1 to 49, wherein the density of the fixed nucleic acid chains is between 10,000 / 100 μm ² and 1,000,000 / 100 μm ² . The nucleic acid chains fixed on the surface may have an anchor or a primer function. In one embodiment, the nucleic acid chains to be analyzed may be complementarily hybridized to the nucleic acid chains fixed on the regenerated surface of the solid phase. In a further embodiment, an enzymatic reaction can be carried out on the primer-template complexes formed. The density of the extension primer capable Matrizenkomplexe is preferably in the following ranges: 1/100 microns ² up to 10/100 .mu.m ² 10/100 to 20/100 microns ² microns ^2, 20/100 to 30/100 microns ² microns ² , 30/100 μm ² to 40/100 μm ² , 40/100 μm ² to 50/100 μm ² , 50/100 μm ² to 60/100 μm ² , 60/100 μm ² to 70/100 μm ² , 70 / 100 μm ² to 80/100 μm ² , 80/100 μm ² to 90/100 μm ² , 90/100 μm ² to 100/100 μm ² , 100/100 μm ² to 120/100 μm ² , 120/100 μm ² to 130/100 μm ² , 130/100 μm ² to 140/100 μm ² , 140/100 μm ² to 150/100 μm ² , 150/100 μm ² to 160/100 μm ² , 160/100 μm ² to 170/100 μm ² , 170/100 μm ² to 180/100 μm ² , 180/100 μm ² to 190/100 μm ² , 190/100 μm ² to 200/100 μm ² , 200/100 μm ² to 250 / 100 μm ² , 250/100 μm ² to 300/100 μm ² , 300/100 μm ² to 400/100 μm ² . In one embodiment, the density of the nucleic acid chains fixed to the regenerated surface of the solid phase is the same over the entire surface of the solid phase. In another embodiment, the prepared solid phase includes regions having different density of the fixed nucleic acid chains. This is particularly advantageous when unknown concentrations of nucleic acid chains to be analyzed are used in a method such as (WO 02088382, WO 02072892).
• 55. Fixed phase with fixed nucleic acid chains according to aspects 1 to 54, wherein a recognizable pattern is attached to the solid phase. For example, this pattern may be a mark that can be recognized by optical means.
• 56. Fixed phase with fixed nucleic acid chains according to aspects 1 to 55, wherein the solid phase forms part of a device for exchanging liquids.
• 57. Fixed phase with fixed nucleic acid chains according to aspects 1 to 56, wherein the solid phase for electromagnetic radiation having wavelengths in the range between 200 nm to 400 nm is permeable.
• 58. Fixed phase with fixed nucleic acid chains according to aspects 1 to 56, wherein the solid phase is permeable to electromagnetic radiation having wavelengths in the range between 200 nm to 2000 nm.
• 59. Fixed phase with fixed nucleic acid chains according to aspects 1 to 56, wherein the solid phase is permeable to electromagnetic radiation having wavelengths in the range between 400 nm to 800 nm. Solid phase can absorb certain areas of the electromagnetic radiation and thus have a filter function.
• 60. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means, wherein the solid phase is used according to aspects 1 to 59.
• 61. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspect 60, wherein labeled components are used.
• 62. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspect 61, wherein the label can be removed from the labeled components by cleavage. The cleavage can be done chemically, photochemically or enzymatically. The cleavage can be carried out, for example, in an aqueous or in an aqueous-organic phase.
• 63. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical agents according to aspects 61 and 62, wherein labeled components include labeled ribonucleoside triphosphates or deoxyribonucleoside triphosphates or dideoxyribonucleoside triphosphates. The naturally occurring nucleosides such as adenosine, guanosine, thymidine, uridine, cytosine as well as their modifications, nucleotide analogs such as 7-deazaadenosine or 7-deazaguanosine can be used. The label may be coupled to the nucleotide at the base, the sugar or the phosphate moiety.
• 64. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical agents according to aspects 61 to 63, wherein labeled components include reversibly labeled ribonucleoside triphosphates or deoxyribonucleoside triphosphates or dideoxyribonucleoside triphosphates.
• 65. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 61 and 62, wherein labeled components include labeled nucleic acids.
• 66. Method for parallel analysis of a plurality of individual nucleic acid molecules by optical means according to aspect 65, wherein labeled components include reversibly labeled nucleic acids.
• 67. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 66, wherein 100 to 10,000 nucleic acid chains are analyzed in parallel
• 68. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 66, wherein parallel 1000 to 100,000 nucleic acid chains are analyzed
• 69. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 66, wherein in parallel 10,000 to 1,000,000 nucleic acid chains are analyzed
• 70. A method for parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 66, wherein in parallel 100,000 to 10,000,000 nucleic acid chains are analyzed
• 71. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 66, wherein parallel 1,000,000 to 100,000,000 nucleic acid chains are analyzed
• 72. A method for parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 66, wherein 100,000,000 to 10,000,000,000 nucleic acid chains are analyzed in parallel
• 73. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 72, wherein events on individual nucleic acid chain molecules are optically detected
• 74. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 73, wherein optical means enable a resolution of up to 0.3 μm
• 75. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 73, wherein optical means allow a resolution of up to 0.5 microns
• 76. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 73, wherein optical means allow a resolution of up to 0.8 microns
• 77. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 73, wherein optical means allow a resolution of up to 1 micron
• 78. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 73, wherein optical means allow a resolution up to 1.2 microns
• 79. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 73, wherein optical means allow a resolution of up to 1.5 microns
• 80. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 73, wherein optical means allow a resolution of up to 2 microns
• 81. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 80, wherein the analysis proceeds by the synthesis of a complementary strand (cyclic sequencing).
• 82. A method of parallel analysis of a plurality of individual nucleic acid molecules with optical means of aspect 81, wherein the analysis is by cyclic sequencing and includes the steps of: 1. providing a solid phase according to aspects 1 to 59 2. binding nucleic acid molecules to be analyzed thereon 3. Performing a cyclic reaction with the solid-phase-fixed nucleic acid chains, including the following steps: 3.1 Incubation of a solution with one or more polymerase and one or more labeled nucleotides (polymerase nucleotide Mixture) with the solid phase under conditions allowing incorporation reaction of nucleotides on the formed primer-template complexes, 3.2 removal of the polymerase-nucleotide mixture and washing of the solid phase 3.3 detection of the signals of incorporated labeled nucleotides, relative coordi If necessary, individual signals can be identified. 3.4 Removal of signals from incorporated nucleotides, 3.5 optionally washing of the solid phase 3.6 optionally repetition of steps 3.1 to 3.5 4. Reconstruction of the sequences of individual nucleic acid molecules from the signals obtained in step 3.3
• 83. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 82, wherein the nucleic acid molecules to be analyzed represent nucleic acid chains with a length between 30 and 300 nucleotides.
• 84. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 82, wherein the nucleic acid molecules to be analyzed nucleic acid chains having a length between 30 and 3000 nucleotides.
• 85. Method for parallel analysis of a plurality of individual nucleic acid molecules by optical means according to aspects 60 to 84, wherein the nucleic acid molecules to be analyzed are deoxyribonucleic acid chains or ribonucleic acid chains.
• 86. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 85, wherein the provided solid phase carries fixed nucleic acids which serve as primers for the sequencing.
• 87. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspect 86, wherein the solid phase provided carries fixed nucleic acids which serve as primers for the sequencing and the nucleic acid molecules to be analyzed are hybridized directly to these primers, with extension-capable primer template Complexes are formed.
• 88. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 85, wherein first the nucleic acid molecules to be analyzed are fixed to the solid phase and then a primer is hybridized to the nucleic acid chains to be analyzed.
• 89. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 82, wherein the fixation of nucleic acid molecules to be analyzed is covalently attached to the solid phase.
• 90. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 82, wherein the fixation of nucleic acid molecules to be analyzed is carried affinely to the solid phase.
• 91. A method for parallel analysis of a plurality of individual nucleic acid molecules with optical means of aspects 82 and 90, wherein the provided solid phase carries fixed nucleic acid chains which serve as anchors for affine fixation of nucleic acid molecules to be analyzed.
• 92. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 82 and 90, wherein the fixation of nucleic acid chains to be analyzed by the hybridization or affine binding to the anchor on the solid phase takes place.
• 93. A method for parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 92, wherein the density of the fixed on the solid phase Extensible primer-template complexes between 5 and 50 complexes per 100 microns ² .
• 94. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 92, wherein the density of the fixed on the solid phase Extensible primer-template complexes between 50 and 250 complexes per 100 microns ² .
• 95. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 94, wherein the nucleic acid molecules to be analyzed are fixed to the solid phase in a random arrangement
• 96. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 94, wherein the nucleic acid molecules to be analyzed are fixed to the solid phase in a predetermined arrangement
• 97. A method of parallel analysis of a plurality of individual nucleic acid molecules with optical means of aspects 82 to 96, wherein nucleotides used in step 3.1 include labeled ribonucleoside triphosphates or deoxyribonucleoside triphosphates or dideoxyribonucleoside triphosphates.
• 98. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 82 to 97, wherein nucleotides used in step 3.1 carry a reversibly terminating group, so that only one labeled nucleotide can be incorporated in an incubation step 3.1.
• 99. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspect 98, wherein after the detection step 3.3, a further step for removing the reversibly terminating group is carried out in, so that the extension reaction can take place in the course.
• 100. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 82 to 99, wherein used nucleotides carry a uniform labeling and in the incubation step 3.1 modified nucleotides of the same base type are added.
• 101. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 82 to 99, wherein used nucleotides carry a base-specific labeling and in the incubation step 3.1 modified nucleotides of different base type are added.
• 102. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 82 to 101, wherein in step 3.3 relative intensity of each signal is determined.
• 103. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 82 to 101, wherein the number of repetitions of steps 3.1 to 3.5 is between 2 and 10.
• 104. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 82 to 101, wherein the number of repetitions of steps 3.1 to 3.5 is between 10 and 25.
• 105. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 82 to 101, wherein the number of repetitions of steps 3.1 to 3.5 is between 25 and 50.
• 105. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 82 to 101, wherein the number of repetitions of steps 3.1 to 3.5 is between 50 and 200.
• 106. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspect 60 to 80, wherein the analysis proceeds by hybridization of complementary labeled nucleic acid chains.
• 107. A method for analyzing nucleic acid chains with the solid phase according to aspects 1 to 59, wherein the nucleic acid chains to be analyzed are fixed to the solid phase and the analysis is carried out by the hybridization of complementary labeled nucleic acid chains.
• 108. A method for analyzing nucleic acid chains with the solid phase according to aspects 1 to 59, wherein nucleic acid strands are fixed in a predefined arrangement on the solid phase and the nucleic acid chains to be analyzed are analyzed by hybridization to the fixed nucleic acid strands.

Abbreviations and term explanations:

Anchor Function

refers on the ability of solid phase-fixed nucleic acid chains, other complementary nucleic acid chains to bind. The process is called hybridization. This bond is counted as affine bonds.

density

Density of the fixed nucleic acid strands or density of the signals: In the representation of the density, an average value of the number of individual elements or events per 100 μm ^{2 is} given in each case.

Production of the surface

manufacturing the surface The solid phase may include several steps. The invention essential Steps of preparation are given. The expert is also other steps known in the processing of surfaces. You will not called explicit if it does not contribute significantly to the invention to have.

incubation

ever according to method, the contact between a solution with labeled components and the surface with fixed nucleic acids differently designated. Some authors call them "incubation steps" others denote they are called "cycle" or "installation cycle".

DNA

Desoxyribonucleic acid different Origin and different length (oligonucleotides, PCR products, Plasmids, genomic DNA, cDNA, ssDNA, dsDNA). RNA - ribonucleic acid

dNTP

2'-deoxy-nucleoside triphosphates, Substrates for DNA polymerases and reverse transcriptases

Marked components

Under labeled components are understood to be molecules that are detectable Wear marker. Preferred are marked in this application Consider nucleotides and labeled nucleic acid chains. The marked Components can also include further modifications.

NTP

Nucleoside triphosphate, Substrates for RNA polymerases

NT

natural Nucleotide, usually dNTP, unless expressly indicated otherwise.

Abbreviation "NT" is also used in the length specification a nucleic acid sequence used, e.g. 1,000 NT. In this case, "NT" stands for nucleoside monophosphates. In the text is abbreviations the majority formed by using the suffix "s", "NT" is for example for "nucleotide", "NTs" stands for several Nucleotides.

NF *

modified Nucleotide, usually dNTP, unless expressly indicated otherwise. NTs * means: modified nucleotides

NSK

Nucleic acid chain.

NACF

Nucleic acid chain fragment. After a fragmentation step, a nucleic acid chain result in several NSKFs.

Plane surface

Surface that preferably has the following features: it allows several signals, preferably more than 100, more preferably more than 1000, with the respective given lens surface distance to detect simultaneously at a lens position.

polymerases

enzymes the complementary one Can incorporate nucleotides into a growing DNA or RNA strand (e.g. DNA polymerases, reverse transcriptases, RNA polymerases)

Primer binding site (PBS)

part the sequence in the NSK or NSKF to which the primer binds.

reference sequence

A already known sequence to which the deviations in the examined Sequence or be determined in the sequences to be examined. As reference sequences can accessible in databases Sequences are used, e.g. from the NCBI database.

Regenerated surface

To the removal of the outer layer The solid phase creates a regenerated surface of the solid phase.

tm

melting temperature

TCEP

Tris-carboxy-ethyl-phosphine

non-specific binding of marked components

Degree of nonspecific binding is expressed as the number of binding events (mean) per 100 μm ² specified. The following gradings are made in this application:
very high unspecific binding: greater than 100/100 μm ²
high unspecific binding: between 50 and 100/100 μm ²
average nonspecific binding: between 20 and 50/100 μm ²
low unspecific binding: between 10 and 20/100 μm ²
very low unspecific binding: below 10/100 μm ²

With increasing density of nonspecific signals increases the probability that the signals from the non-specific events with the signals overlap by specific events and visually indistinguishable. The extent of unspecific Bonding to the surface, that makes the analysis of processes at the single-molecule level difficult to impossible, depends strongly from the resolution of the detection system and abilities subsequent systems, such as image recognition systems (e.g. Camera, software) to distinguish adjacent signals. The introduction the non-specific binding steps were performed for a resolution of 0.25 μm to 0.4 μm.

Wide-field optics

Under Widefield optics are projected detection devices that a resolution in the range of over 0.2 μm, e.g. a resolution of 0.5 μm or 1 μm. This generic term distinguishes itself from the near-field optics, the one resolution in the range of a few nanometers.

Of the Term limits the relevant detection methods are not. For example can do the following Detection types are used: wide-field fluorescence microscope (epifluorescence), Laser scanning microscope (epifluorescence) or a TIRF microscope (Total Internal Reflection Fluorescence Microscope).

For the detection The fluorescence signals of individual molecules are different variants the construction of such an apparatus is possible (Weston et al., J. Chem. Phys. 1998, p.109 p. 7474, Trabesinger et al. Anal. Chem. 1999 v.71 p.279, Adachi et al. Journal of Microscopy 1999 v.195 p.125, Unger et al. BioTechniques 1999 v.27 p.1008, Ishijaima et al. Cell 1998 v.92 P.161, Dickson et al. Science 1996, v. 74 p. 966, Tokunaga et al. Bichem.Biophys.Res.Com. 1997 v. 235 p. 47, "Confocal Laser Scanning Microscopy" 1997 Ed. Sheppard, BIOS Scientific Publishers, "New Techniques of optical microscopy and microspectroscopy 1991 Ed. R. Cherry CRC Press, Inc., "Fluorescence microscopy "1998 2.ed. Herman BIOS Scientific Publishers, "Handbook of biological confocal microscopy" 1995 J. Pawley plenum Press). Differences in their concrete structure arise the variation of their individual parts. The device for the excitation light can e.g. based on a laser, a lamp or diodes function. For the detection device can Both CCD cameras, line scan cameras or as well as photomultiple tube (PMT) or diodes are used. Other examples of technical details see ("Confocal laser Scanning Microscopy "1997 Ed. Sheppard, BIOS Scientific Publishers, "New Techniques of Optical Microscopy and microspectroscopy "1991 Ed. R. Cherry CRC Press, Inc., "Fluorescence microscopy "1998 2.ed. Herman BIOS Scientific Publishers, "Handbook of biological confocal microscopy" 1995 J. Pawley plenum Press).

Especially combinations of the surfaces according to the invention with a wide field optic are advantageous a local one Resolution, which lies in one of the following ranges: between 0.2 μm to 0.3 μm, 0.3 μm to 0.4 μm, 0.4 μm to 0.5 μm, 0.5 μm to 0.6 μm, 0 , 6 μm to 0.7 μm, 0.7 μm to 0.8 μm, 0.8 μm to 0.9 μm, 0.9 μm to 1 μm, 1 μm to 1.5 μm, 1.5 μm to 2 μm.

Detailed description the invention:

• 1. Bereitstellung einer festen Phase
• 2. Bindung von zu analysierenden Nukleinsäureketten an die feste Phase unter Ausbildung extensionsfähiger Primer-Matrize-Komplexe,
• 3. Durchführung einer zyklischen Reaktion mit den auf der festen Phase fixierten Primer-Matrize-Komplexen, die folgende Schritte einschließt: 3.1 polymerasenabhängiger Einbau von markierten Nukleotiden in die gebildeten Primer-Matrize-Komplexen, 3.2 Waschen der festen Phase 3.3 Detektion der Markierung von eingebauten markierten Nukleotiden, wobei relative Koordinaten einzelner Signale identifiziert werden 3.4 Entfernung der Signale von eingebauten Nukleotiden, 3.5 Waschen der festen Phase 3.6 gegebenenfalls Wiederholung der Schritte 3.1 bis 3.5
• 4. Rekonstruktion der Sequenzen von einzelnen Nukleinsäureketten aus den unter Schritt 3.3 erhaltenen Signalen

In modern analytics, methods are increasingly being used that are highly sensitive. Representative of such methods are considered in this application methods based on the analysis of individual molecules. They make ultimate demands on the materials used, for example, the strength of the background signal. Methods such as (WO 02088382, WO 02072892, WO 0070073) are based on the optical detection of the events (for example incorporation of labeled nucleotides into the nucleic acid chains) which take place at single-molecule level (eg fluorescence signals from individual labeled incorporated nucleotides are detected). For example, such a method includes the following steps:

• 1. Providing a solid phase
• 2. binding of nucleic acid chains to be analyzed to the solid phase to form expandable primer-template complexes,
• 3. Carrying out a cyclic reaction with the solid phase-fixed primer-template complexes, which includes the following steps: 3.1 polymerase-dependent incorporation of labeled nucleotides into the formed primer-template complexes, 3.2 washing of the solid phase 3.3 detection of the label of incorporated labeled nucleotides, whereby relative coordinates of individual signals are identified 3.4 Removal of signals from incorporated nucleotides, 3.5 washing of the solid phase 3.6 if necessary repetition of steps 3.1 to 3.5
• 4. Reconstruction of the sequences of individual nucleic acid chains from the signals obtained in step 3.3

at such methods will become common a high concentration of labeled reagents used with it enzymatic reactions can proceed in step 3.1. repeated Contact between marked components and the surface of the solid phase leads to their unspecific binding to the surface. This non-specific binding from labeled constituents to the surface provides an important source for the background signal represents.

There the analysis is based on the detection of fluorescence from single molecules, the spectral and geometric properties differ the signals of incorporated nucleotides and signals of nonspecific to the surface bound, labeled nucleotides barely. At a great density nonspecifically bound signals overlap the specific ones and the non-specific signals, resulting in a faulty analysis leads.

For the sequencing method optical means are used which have a resolution of 0.2 microns to 2 microns (WO 03031947). Such optical means are commonly referred to as "wide field optics". The fortune of Apparatus, the signals of individual molecules as individual signals to distinguish depends on the density of signals on the surface and the resolution of the Equipment off: With decreasing resolution the detection apparatus sinks the assets, close together To distinguish signals; increases with increasing density with permanent resolution the probability that the signals from the detection apparatus as overlapping be detected. At a large density of unspecific bound labeled components is also the probability a random overlap of specific and nonspecific signals. Thus, there is a great need surfaces with one as possible to develop low non-specific binding.

For example, at a resolution of 0.3 μm and a density of 40 to 60 specific signals per 100 μm ² , it is desirable that the density of the non-specifically bound signals be less than 20 per 100 μm ² over the duration of the analysis (eg 100 cycles) is. This allows a good distinction between specific and nonspecific signals.

A low unspecific binding of labeled components is particular then important if multiple incubation step with labeled nucleotides he wishes are, for example, 5 to 100 steps or even up to 500 steps.

Problem of development a surface with low unspecific binding:

There the building blocks of the nucleic acids, the nucleotides include both hydrophilic and hydrophobic moieties, It is not surprising that they are attached to surfaces of different materials unspecific to a certain extent tie. The dye-modified nucleotides have more potential sites that can lead to binding to the surface. These Facts usually make the development of a surface of the solid phase with a low non-specific binding of labeled Components especially difficult.

Several variants of surface preparation or process design have already been proposed which result in a reduced background signal due to the non-specific binding of labeled components lead.

State of the art

In In the application WO 02072892 a surface was described which reduced one Binding of labeled nucleotides compared to standard surfaces has: at concentrations of up to 0.1 μmol / L of labeled nucleotides It is possible to perform up to 3 incubation cycles until the density of the signals from unspecific to the surface bound labeled nucleotides no clear analysis of the incorporation events on individual molecules allowed with a wide field optics. To further installation events of to distinguish the nonspecifically bound nucleotides set the authors FRET as a means of stimulating labeled nucleotides a procedure, resulting in analysis only at short distances of a few Nanometers allows and were thus able to detect up to 6 installation events. Similar Results are also achieved with other known surface.

It is known in the art that nucleic acids by binding to silica can be isolated (boom et al., 1990; Boom et al., 1999; Chiu et al., 2003; Dederich et al., 2002; Diehl et al., 2001; Dolan et al., 2001; Hackler et al. 2003; Maitra and Thakur, 1994; Marko et al., 1982; Merkelbach et al., 1997; Miller et al., 1999; Mygind et al., 2003; Smith et al., 1995; Vogelstein and Gillespie, 1979; Zeillinger et al., 1993). This is due to the fact that nucleic acids are capable of silica nonspecifically bind, using both long and short nucleic acids Can bind silica. This is one of the reasons why glass-based DNA chip a high degree of non-specific binding of nucleic acids have ("DNA Microarrays, "Bowtell, 2003, ISBN 0-87969-624-9, "Microarray-Biochip Technology" M Schena, 2000, ISBN 1-881299-37-6).

The surface of the present invention is the surface of a solid phase including SiO ₂ as a main component. Surprisingly, it was possible to prepare and modify the glass surface so that the non-specific binding of labeled nucleotides and labeled nucleic acids was drastically reduced. A subsequent chemical modification of the surfaces for coupling the nucleic acids does not lead to a significant increase in the unspecific binding of labeled and unlabeled nucleic acids.

The Surface of the invention solid phase is characterized by a much lower nonspecific Binding of labeled nucleotides and labeled nucleic acid chains in comparison to the prior art. This property leads to a better discrimination of the specific signal over the nonspecific signal and brings many advantages in their application in highly sensitive procedures.

Furthermore Furthermore, the surface of the invention is characterized by a low nonspecific Binding on repeated incubation steps of labeled components with the surface out. The surface according to the invention can thus in processes with both low and large numbers be used at incubation steps.

In a preferred embodiment becomes the surface used in procedures where the number of incubation steps with marked components in the following ranges: 1 to 10 Incubation steps, 5 to 10 incubation steps, 10 to 20 incubation steps, 20 to 50 incubation steps, 50 to 100 incubation steps, 100 up to 200 incubation steps, 200 to 500 incubation steps, 500 up to 1000 incubation steps.

marked Components can wear different markers. For example, they can with fluorescent dyes be marked, such as rhodamines and their derivatives (rhodamine 110, tetramethylrhodamine, rhodamine 6G), cyanine dyes (e.g., Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, (Amersham-Pharmacia, Freiburg, Germany), Alexa Dyes (e.g., Alexa 546, Alexa 555, Alexa 568, Alexa 590 (Molecular Probes Europe BV, Leiden, The Netherlands as Molecular Probes), Atto dyes (Atto-Tec GmbH, Siegen, Germany).

there can one or more dyes are bound per one labeled component be, e.g. multiple dyes per one labeled oligonucleotide.

marked Components can dissolved in different buffers such as acetate, glycine, phosphate, carbonate, Borate, Tris buffer. The pH is preferably in the range between 6 and 10.

Examples of labeled components are nucleotides (ribonucleoside triphosphates, 2'-deoxyribonu kleoside triphosphates, 2 ', 3'-dideoxyribonucleoside triphosphates) such as dUTP-Cy3, dCTP-Cy3, dUTP-fluorescein, dUTP-Alexa 555, dUTP-rhodamine. Many other modifications are commercially available (Molecular Probes, Inc., Amersham Bioscience, Sigma, NEN-Perkin Elmer Inc.). Labeling is often coupled to the linker Hobbs et al. US Pat. No. 5,047,519 or other linkers, eg Klevan US Pat. No. 4,828,979, Seela US Pat. No. 6211158, US pat. No. 4804748, EP 0286028 , Hanna M. Method in Enzymology 1996, v.274, p.403, Zhu et al. NAR 1994 v.22 p.3418, Jameson et al. Method in Enzymology, 1997, v. 278, p. 363-, Held et al. Nucleic acid research, 2002, v. 30, 3857, Held et al. Nucleosides, nucleotides & nucleic acids, 2003, v. 22, p. 391, Short US Pat. No. 6579704, Odedra WO 0192284, Odedra WO 03048178, Herrlein et al. Helvetica Chimica Acta, 1994, V. 77, p. 586, Canard US. Pat. No. 5,798,210, Kwiatkowski US Pat. No. 6,254,575, Kwiatkowski WO 01/25247, Parce WO 0050642, Faulstich et al. DE 4418691 , Dissertation "Synthesis of base - modified nucleoside triphosphates and their enzymatic polymerization to functionalized DNA", Oliver Thum, Bonn 2002.

In another embodiment, the label may be cleaved from the nucleotide under mild conditions. Many examples are known for such nucleotides Tcherkassov WO 02088382, Short US Pat. No. 6579704, Odedra WO 0192284, Odedra WO 03048178, Canard US Pat. No. 5,798,210, Kwiatkowski US Pat. No. 6,254,475, Kwiatkowski WO 01/25247, Parce WO 0050642, Faulstich et al. DE 4418691 , Ju et al. WO 02/29003, Milton et al. WO 2004/018493, Milton et al. WO 2004/018497,

marked nucleic acids like oligonucleotides can be purchased commercially (Sigma-Aldrich-Fluka (Taufkirchen, Germany) hereinafter referred to as Sigma, MWG-Biotech, Ebersberg near Munich, Germany, (hereinafter referred to as MWG Biotech). The labeling of longer nucleic acids like cDNA or plasmid DNA can be made by known methods ("Molecular cloning" 1989, Ed. Maniatis, Cold Spring Harbor Laboratory).

in the Furthermore, the inventive surface of the solid phase is characterized from that nucleic acid chains to this surface covalently bound and the surface nevertheless a low nonspecific binding of labeled components having.

The density of nucleic acid chains bound to the surface of the solid phase can vary. In a preferred embodiment, the density of the fixed nucleic acid chains in one of the following areas (note: the number of nucleic acid strands per 100 microns ^2): 0.1 - 1/100 microns ^2, 1 - 10/100 microns ^2, 10 - 20/100 μm ² , 20 - 30/100 μm ² , 30 - 40/100 μm ² , 40 - 50/100 μm ² , 50 - 60/100 μm ² , 60 - 70/100 μm ² , 70 - 80/100 μm ² , 80 - 90/100 μm ² , 90 - 100/100 μm ² , 110 - 120/100 μm ² , 120 - 130/100 μm ² , 130 - 140/100 μm ² , 140 - 150/100 μm ² , 150 - 160/100 microns ^2, 160 - 170/100 microns ^2, 170 - 180/100 microns ^2, 180 - 190/100 micron ^2, 190 - 200/100 microns ^2, 200 - 220/100 microns ^2, 220 - 240 / 100 μm ² , 240 - 260/100 μm ² , 260 - 280/100 μm ² , 280 - 300/100 μm ² , 300 - 350/100 μm ² , 350 - 400/100 μm ² , 400 - 450/100 μm ² , 450 - 500/100 μm ² , 500 - 550/100 μm ² , 550 - 600/100 μm ² , 600 - 700/100 μm ² , 700 - 800/100 μm ² , 1 - 50/100 μm ² , 1 - 100/100 μm ² , 10 - 1 00/100 μm ² , 50-200 / 100 μm ² , 50-500 / 100 μm ² .

² 1 1/100 microns, - - 10/100 microns 0.1: In another embodiment, the density of the extentionsfähigen primer Matrizenkomplexe is located in one or more of the following areas (specification: number of extension capable primer Matrizenkomplexe per 100 microns ^{2) 2,} 10 - 20/100 microns ^2, 20 - 30/100 microns ^2, 30 - 40/100 microns ^2, 40 - 50/100 microns ^2, 50 - 60/100 microns ^2, 60 - 70/100 microns ^2, 70 - 80/100 μm ² , 80 - 90/100 μm ² , 90 - 100/100 μm ² , 110 - 120/100 μm ² , 120 - 130/100 μm ² , 130 - 140/100 μm ² , 140 - 150/100 μm ² , 150 - 160/100 μm ² , 160 - 170/100 μm ² , 170 - 180/100 μm ² , 180 - 190/100 μm ² , 190 - 200/100 μm ² , 200 - 220 / 100 microns ^2, 220 - 240/100 microns ^2, 240 - 260/100 micron ^2, 260 - 280/100 microns ^2, 280 - 300/100 microns ^2, 300 - 350/100 microns ^2, 350 - 400/100 microns ² , 400 - 450/100 μm ² , 450 - 500/100 μm ² , 500 - 550/100 μm ² , 550 - 600/100 μm ² , 600 - 700/100 μm ² , 700 - 800/100 μm ² , 1 - 50/100 μm ² , 1 - 100/100 μm ² , 10 - 100/100 μm ² , 50 - 200/100 μm ² , 50 - 500/100 μm ² .

at a random one Distribution of primer-template complexes on the surface can a certain proportion of the population as individual sequencing sites be differentiated from the adjacent sequencing sites. The Size of the share of the population in which signals from incorporated nucleotides on primer-template complexes can be recognized individually i.e. without overlap with the signals from the neighboring places depends essentially on the Density of the fixed and extendible primer-template complexes and from the resolution the detection apparatus. Example of the calculation of units primer-template complexes, without overlap to be detected with the neighboring sites are in the table 1 to 8 indicated. In this embodiment, only extension-capable primer template complexes considered.

It can be seen clearly that the relative proportion of the individually recognized primer-template complexes in a low density of the primer-template complexes on the surface is large. With increasing density or with decreasing resolution, the relative proportion of individually recognized events on primer-template complexes decreases as more and more sites overlap with the neighboring sites and can not be distinguished by the apparatus as events on individual primer-template complexes.

For sequencing the nucleic acid strands with a method such as (WO 02088382, WO 03020968) it is not necessary that the signals from all participating in the reaction primer-template complexes as single molecule signals be differentiated. It is sufficient, if events from only part of the population involved in the sequencing reaction participates as events on individual primer-template complexes become.

When Events can in this embodiment For example, incorporation of labeled nucleotides or cleavage the mark can be defined.

In a preferred embodiment The application will combinations between the surface of the invention and the detection apparatus used in which the proportion of the population primer-template complexes where events are individually (i.e. delimitable from the events on adjacent primer-template complexes) be detected in the following ranges: 5% to 10%, 10% up to 15%, 15% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% up to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90 to 95%.

Examples for connections between the resolution of the optical system, the density of the extensible primer nucleic acid complexes and the percentage of the total population of signals from built-in nucleotides that can be individually recognized are in Table 1 to 8. In these tables will be results a computer simulation for the influence of the dissolution the detection system and the density of the extensible primer nucleic acid (referred to as "sites", an abbreviation for sequencing sites) on the degree of overlap the neighboring places presented. The degree of overlap is recognized as a percentage of sites that are considered individual be reproduced.

Table 1, resolution 0.2 μm

At a resolution of 0.2 microns, the preferred actual density is between 40 and 1300 sites per 100 microns ² , in another embodiment, the preferred density is between 100 and 600 sites per square inch 100μm ² .

Table 2, resolution 0.3 μm

At a resolution of 0.3 μm, the preferred actual density is between 30 and 500 spots per 100 μm ² , in another embodiment the preferred density is between 70 and 300 spots per 100 μm ² .

In 2 Examples of different signal densities are given, which were recorded with a resolution of 0.3 μm.

Table 3, resolution 0.4 μm

At a resolution of 0.4 μm, the preferred actual density is between 10 and 250 spots per 100 μm ² , in another embodiment the preferred density is between 30 and 200 spots per 100 μm ² .

Table 4, resolution 0.5 μm

At a resolution of 0.5 μm, the preferred actual density is between 7 and 200 sites per 100 μm ² , in another embodiment the preferred density is between 30 and 150 sites per 100 μm ² .

Table 5, resolution 0.6 μm

At a resolution of 0.6 μm, the preferred actual density is between 5 and 100 sites per 100 μm ² , in another embodiment the preferred density is between 20 and 80 sites per 100 μm ² .

Table 6, resolution 0.8 μm

At a resolution of 0.8 microns, the preferred actual density is between 5 and 50 points per 100 microns ^2, in another embodiment, the preferred density is between 10 and 40 characters per 100 microns. ²

Table 7, resolution 1 μm

At a resolution of 1 μm, the preferred actual density is between 5 and 50 spots per 100 μm ² , in another embodiment the preferred density is between 10 and 30 spots per 100 μm ² .

Table 8, resolution 2 μm

At a resolution of 2 μm, the preferred actual density is between 2 and 10 places per 100 μm ² .

As can be seen from Tables 1 to 8, play the resolution and the density of passages (where passages in the broadest sense are independent, random the surface distributed events) play a major role in the question of how big the Proportion of individual, i. independently from the neighboring sites detectable events in the total population is.

in the In connection with these examples, the importance of the surface according to the invention with a very low non-specific binding of labeled components even clearer. At low numbers of nonspecifically bound Components increases the yield of sequences of primer-template complexes, which can be evaluated after a sequencing reaction. For the detection can Detection devices are used, which have a lower resolution.

Furthermore, the surface of the solid phase according to the invention is distinguished by the fact that many enzymes, such as polymerases and ligases, recognize the nucleic acid chains fixed on the surface as substrates. For example, the following polymerases are suitable for the enzymatic reaction:
DNA-dependent DNA polymerases, DNA-dependent RNA polymerases, RNA-dependent DNA polymerases, RNA-dependent RNA polymerases.

In the choice of polymerase, the type of fixed nucleic acid to be analyzed (RNA or DNA) plays a decisive role:
If RNA is used as substrate (eg mRNA) in the sequencing reaction, commercial RNA-dependent DNA polymerases can be used, eg AMV reverse transcriptase (Sigma), M-MLV reverse transcriptase (Sigma), HIV reverse transcriptase without RNAse- Activity. For certain applications, reverse transcriptases may be largely free of RNAse activity ("Molecular cloning" 1989, Ed. Maniatis, Cold Spring Harbor Laboratory), eg in mRNA migation for hybridization experiments.

If DNA is used as substrate (eg cDNA), all DNA-dependent DNA polymerases with or without 3'-5 'exonuclease activity (DNA replication "1992 Ed A.Kornberg, Freeman and company NY ), eg, modified Sequenase Version 2 T7 polymerase (Amersham Pharmacia Biotech), Klenow fragment of DNA polymerase I with or without 3'-5 'exonuclease activity (Amersham Pharmacia Biotech), polymerase beta of various origins (Animal Cell DNA polyme rases "1983, Fry M., CRC Press Inc., commercially available from Chimerx) thermostable polymerases such as Taq polymerase (GibcoBRL), proHA DNA polymerase (Eurogentec), Vent, Vent exo minus, Pfu polymerase, Pwo- Polymerase, Tli polymerase DNA-dependent RNA polymerases can also be used, for example E. coli RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase.

In The registration will be DNA-dependent DNA polymerases as examples of considered all polymerases.

The property of the surface, nonspecific binding of labeled and unlabelled nucleotides and nucleic acid only to a small extent, as well as the ability to specifically bind nucleic acids to the surface according to the invention, offers an overall great advantage over existing surfaces and allows the use of such surfaces in biosensors for conducting experiments with labeled components such as a substrate for DNA-chip technology ("DNA Microarrays" Ed., by D. Bowtell & J. Sambrook, 2003, CSHL Press, ISBN: 0-87969-625-7). In particular, the solid phase surface according to the invention is suitable for analytical methods with incorporation of labeled nucleotides into the nucleic acids coupled to a solid phase, such as mini-sequencing (Suomalainen A et al., Methods Mol Biol. 2003; 226: 361-6, Liljedahl U et al Pharmacogenetics, 2003 Jan; 13 (1): 7-17, Olsson C et al., Methods Mol Biol. 2003; 212: 167-76), primer extension (Pirrung MC et al., Bioorg Med Chem Lett., 2001, Sep. ; 11 (18): 2437-40, Cai H, et al., Genomics 2000 Jun 1; 66 (2): 135-43, Kurg A et al., Genet Test., 2000; 4 (1): 1-7 , Pastinen T et al., Genome Res. 1997 Jun; 7 (6): 606-14). U.S. Pat. No. 6,287,766, U.S. Pat. No. 2003148284, U.S. Pat. No. 2003082613, U.S. Pat. EP 1256632 , WO0194639. Single-Molecular Sequencing (Tcherkassov WO 02088382, Seeger WO 0018956, Kartalov WO 02072892).

in the Another is the use of such a surface on the example of highly parallel Sequencing of individual nucleic acid molecules shown.

material

The surface according to the invention represents the surface of a solid phase. The solid phase preferably consists of SiO ₂ (silica) as main component. In this case, silica may be present as glass or quartz.

In one embodiment, the proportion of SiO ₂ in the solid phase is between 10 and 50%. In another embodiment, the solid phase is more than 50% SiO ₂ , in another embodiment the solid phase is more than 70% SiO ₂ , in another embodiment the solid phase is more than 90% SiO ₂ , in one In another embodiment, the solid phase is more than 95% SiO ₂ , in another embodiment the solid phase is more than 99% SiO ₂ .

In a further embodiment of the invention, the solid phase consists of silicon, the surface of the solid phase having a layer of SiO ₂ . The chemical modifications described in this application relate to the changes and modifications of this SiO ₂ layer.

In addition, glass serves as an example of the solid phase which has an SiO ₂ surface. Different types of glass can be used, for example borosilicate glass. The suitable material is commercially available, for example Menzel coverslips and slides (Omnilab Laboratory Center GmbH, Bremen, Germany). Preferably, the solid phase material has few or no fluorescent components.

In an embodiment is the surface the solid phase plan.

In a further embodiment is the solid phase for the electromagnetic radiation is transparent in the following areas: 200 nm to 500 nm, 200 nm to 800 nm, 400 nm to 700 nm, 400 nm up to 1000 nm, 500 nm to 2000 nm.

In an embodiment Only the solid phase is used as material in the manufacturing process. The integration of the surface the solid phase into a device for exchanging the solutions, e.g. into a reagent vessel or a micro-channel device, takes place accordingly only after the Production of the surface according to the invention.

In another embodiment, the solid phase is a component of a reagent vessel, a microchannel device or an array of reagent vessels, for example a microtiter plate and the surface is prepared directly in such a reagent vessel or microchannel. Different design possibilities for microfluidic channels are known to the person skilled in the art (eg "Integrated microfabicated biodevices" 2002 ISBN 0-8247-0606-4).

In another embodiment can individual steps of making the surface at different stages the assembly of reagent vessels and Microchannel devices take place.

In a further embodiment includes a microchannel device has two or more separate components which have the properties of the solid phase surface according to the invention.

In a further embodiment is a protective layer (a mask) on the surface of the solid phase, so that the machining processes only certain Areas of the fixed phase. This mask can be, for example consist of a photosensitive material and then by Light exposure are removed. Examples of such masks are the Specialist known.

Production, general:

The Production of a surface for the Method for highly parallel sequencing of individual nucleic acid chains essentially involves a combination of a removal step the outer layer of the solid phase by a chemical or physical action and a coupling of nucleic acids this surface.

The Removal of the outer layer the solid phase leads to a drastic reduction of nonspecific binding of nucleotides and nucleic acids to the newly created surface the solid phase, s. Example 1. After the removal of the outer layer creates a surface the solid phase, referred to in this application as a "regenerated surface".

• 1. Entfernung der Außenschicht der festen Phase, wobei eine regenerierte Oberfläche der festen Phase entsteht, die eine SiO₂ Oberfläche ist.
• 2. Kopplung bzw. Synthese von Nukleinsäureketten an die regenerierte Oberfläche der festen Phase
• 3. gegebenenfalls Kopplung weiterer Substanzen

The production of the surface in one embodiment includes the following essential steps:

• 1. Removal of the outer layer of the solid phase, wherein a regenerated surface of the solid phase is formed, which is a SiO ₂ surface.
• 2. Coupling or synthesis of nucleic acid chains to the regenerated surface of the solid phase
• 3. optionally coupling other substances

• 1. Entfernung der Außenschicht der festen Phase, wobei eine regenerierte Oberfläche der festen Phase entsteht, die eine SiO₂ Oberfläche ist.
• 2. Kopplung bzw. Synthese einer Linker-Komponente an die regenerierte Oberfläche der festen Phase,
• 3. Kopplung bzw. Synthese von Nukleinsäureketten an die/an der Linker-Komponente.
• 4. gegebenenfalls Kopplung weiterer Substanzen

In a further embodiment, the preparation includes the following essential steps:

• 1. Removal of the outer layer of the solid phase, wherein a regenerated surface of the solid phase is formed, which is a SiO ₂ surface.
• 2. coupling or synthesis of a linker component to the regenerated surface of the solid phase,
• 3. Coupling or Synthesis of Nucleic Acid Chains to / on the Linker Component.
• 4. optionally coupling other substances

The Removal of the outer layer takes place by chemical reaction in an aqueous or aqueous-organic Solution. In one embodiment becomes the regenerated surface not in the further course of the manufacturing and analysis process dried out. These steps can be integrated into a manufacturing process, which includes more Includes steps for editing the surface.

Different substances can be coupled to the regenerated surface of the solid phase. Many examples of couplings to a glass or SiO ₂ surface are known ("Silane Coupling Agents" 2 Ed. 1991, ISBNO-306-43473-3, "DNA Microarrays", Bowtell, 2003, ISBN 0-87969- 624-9, "Microarray-Biochip Technology" M Schena, 2000, ISBN 1-881299-37-6, Kumar et al Nucleic Acid Research, 2000/28, e71, Guo et al., Nucleic Acid Research, 1994, p. 22, 5456, Lindroos et al Nucleic Acid Research, 2001 v. 29, No. 13, e69, Taylor et al., Nucleic Acid Research, 2003 v. 31, e87,).

In an embodiment be substances, such as nucleic acid chains or their building blocks, Nucleotides, proteins, such as streptavidin or antibodies, linear and branched polymers such as PEG, polyphosphates, polyacrylates, polyacrylamides (cross-linked and not cross-linked), dendrimers coupled directly to the surface become.

In another embodiment, linker molecules may be introduced between the above-mentioned substances and the regenerated surface. These linkers can carry reactive groups that serve to couple substances, such as epoxy, aldehyde, carboxy ("Silane Cou Pling Agents, 2nd Ed., 1991, ISBNO-306-43473-3, "DNA Microarrays," Bowtell, 2003, ISBN 0-87969-624-9, "Microarray-Biochip Technology" M. Schena, 2000, ISBN 1-881299 Nucleic Acid Research, 2000, 28, e71, Guo et al., Nucleic Acid Research, 1994 v. 22, 5456, Lindroos et al., Nucleic Acid Research, 2001, v. 29, No. 37-6, Kumar et al 13, e69, Taylor et al Nucleic Acid Research, 2003 v. 31, e87,).

At the regenerated surface can uniform substance populations, such as uniform Protein molecules, e.g. Streptavidin, or uniform nucleic acid populations, such as Oligo nucleotides or PCR products, as well as mixed populations of molecules, such as cDNA, fragmented genomic DNA become.

At the surface can simultaneously or consecutively also different substance classes be bound, for example proteins, nucleic acids, polymers such as PEG, polyacrylamide, polyphosphates.

Surprisingly has the surface according to the invention despite further modifications generally a very weak non-specific Binding behavior opposite different labeled nucleotides and nucleic acid chains. This behavior has been described in several, in molecular biology and biochemistry in common use Buffer systems, such as Tris-HCl, phosphate buffer and Borate buffer over wide pH ranges, preferably between 7 and 11, and concentration ranges detected by buffer substances. The concentration of the buffer solutions is preferably in the following ranges: between 5 and 50 mmol / l, between 20 and 200 mmol / l, between 100 and 1000 mmol / l.

in the Another is the coupling of nucleic acid chains to the regenerated surface considered closer and serves as an example for further coupling options other substances.

The Coupling of nucleic acids is preferably covalent or affine. Many coupling methods for covalent Couplings are known to those skilled in the art ("Silane Coupling Agents" 2 Ed. 1991, ISBNO-306-43473-3, "DNA Microarrays", Bowtell, 2003). ISBN 0-87969-624-9, "Microarray biochip Technology "M Schena, 2000, ISBN 1-881299-37-6, Kumar et al. Nucleic Acid Research, 2000 v. 28, e71, Guo et al. Nucleic Acid Research, 1994 BC. 22, 5456, Lindroos et al. Nucleic Acid Research, 2001 b. 29, No. 13, e69, Taylor et al. Nucleic Acid Research, 2003 v. Chr. 31, e87,). Examples for affine Couplings are couplings over Biotin-streptavidin linkages or coupling by hybridization of complementary Nucleic acid strands. nucleic acid chains can also directly on the surface synthesized (Capaldi et al., 2000; Damha et al., 1990; Devivar et al., 1999; Habus et al., 1998; Pon, 1993; Pon et al. 1988; Sinha, 1993; Song et al., 2003; Veiko et al., 1991; Vu et al., 1990) or (US Pat. No. 5,753,788, US Pat. No. 5,831,070, US Pat. No. 5919523, US Pat. No. 6022963, US Pat. No. 6147205, US Pat. 6153743, US Pat. No. 6262216, US Pat. No. 6310189, US Pat. No. 6480324, US Pat. US Pat. No. 6486287).

In an embodiment may be the coupling of nucleic acid chains done in a particular arrangement. In another embodiment be nucleic acids in random Way to the surface bound (WO 02088382).

At the surface of the invention can in different stages of manufacturing patterns (e.g., adjustment pattern or "Landmarker", WO 02088382, WO 03031947) are applied. For example, microparticles with a Diameter of a few microns are bound to the surface. These particles preferably do not bind labeled nucleotides or nucleic acids.

In a preferred embodiment can these particles are made visible. For example, absorb these particles are electromagnetic radiation of a certain wavelength or have fluorescent properties. In a further embodiment can they are made visible by light scattering. Example of such Particles form ink particles or polysterene beads represents.

The inventive surface was for the Analyzes are developed in which labeled nucleotides or nucleic acid chains be used. These components contribute to buffer solutions usually a negative charge and can be caused by electrostatic Bind binding to different surfaces. For this reason it is not advantageous to apply positive charges to the surface according to the invention. For example, coupling of amino groups to the surface can result in formation contribute to positive charges. Such treatments are the surface not beneficial. For example, treatments with aminopropyltrimethoxysilanes or polylysine to a strong binding of nucleic acids and Nucleotides to the surface.

Examples:

Examples should serve to illustrate the invention and not to limit.

The Production of the surfaces is exemplified by the commercially available cover glasses and slides for microscopy shown. These glasses are normally pre-cleaned and can be kept dry (Menzel cover glasses and slides, from Omnilab Laboratory Center, Bremen, Germany).

The solid phase, which served as material for the preparation of the surface, was used as part of a reagent vial. The reagent vial was in an execution as a microfluidic channel in front. In another version were microtiter plates with a flat glass bottom (e.g., company Greiner Bio-One GmbH, Frickenhausen, Germany or Molecular Machines & Industries GmbH, Glattburg, Schweitz). The results of the production the surface were substantially similar in both types of reagent tubes. The examples are for the surface the solid phase presented as part of a microfluidic channel served.

Microfluidic channel (MFC)

The microfluidic channels ( 3a ) were assembled as follows: From Parafilm (Merck, Darmstadt) a mask is cut out. This mask is placed wrinkle-free on a slide. The slide (Menzel) with the mask is heated to 130 ° C for 30 sec, resulting in the melting of the parafilm, and then cooled to RT, resulting in the solidification of the parafilm. The coverslip (Menzel) is placed on the mask and lightly pressed. The slide with the mask and the coverslip is now heated to 120 ° C, which again leads to the melting of the parafilm. In this way, slide and the cover glass are connected to each other, wherein in the middle of a microchannel is formed with a constant height of about 0.2 mm. Through the micro-channel, the exchange of solutions ( 3b ). The volume of the microchannel is 10 μl.

in the Microfluidic channel can the liquid be replaced. This exchange allows different reagents as solutions be used in contact with the solid phase and in sequential Steps are exchanged. If necessary, a microchannel was made several times washed by a larger total volume a solution was replaced by the microchannel.

detection:

The basic structure of the apparatus used for single-molecule microscopy is shown schematically in FIG 4a and is shown in detail in patent applications WO 03031947, WO 02088382.

In this work a wide field fluorescence microscope Axioplan 2 (Zeiss, Oberkochen, Germany) with mercury vapor lamp HBO 100, objective Planneofluar 100x, NA 1.4 (Zeiss), camera AxioCam (Zeiss) and computer with software for control and analysis was used. Images are taken from the surface as in 4b shown. Exposure time was 7 sec. Each image reflects the two-dimensional distribution of the signals picked up by the camera's CCD (1388 × 1040 pixels). In this case, a recording represents a range of 60 μm × 80 μm of the surface. The signals of individual molecules were distinguished from the background noise (FIG. 5 ). Its apparent diameter was about 6 pixels or about 300 nm. The total resolution of the system was 0.3 μm.

An alignment pattern or landmark was applied to the surface of the solid phase. For example, a dilute ink solution (Pelickan AG, Hannover, Germany) was brought into contact with the surface in phosphate buffer. The ink particles (their apparent diameter about 0.3 to 2 μm) bound to the surface and could be visualized with a microscope (eg with transmitted light). The binding of the ink particles took place at random locations of the surface. The density of the binding of the ink can vary and was, for example, 3 to 100 particles per 100 μm ² , these particles are to be seen as dark areas on the fluorescence images, eg in 1A , B, C, D, 7A . 8A , For each area of the surface, specific patterns of ink particles were created. Since these ponds are bound to the surface and maintain their position, they can be used to find the same positions and for adjustment (WO 03031947, WO 02088382).

Further description of the processing of the surface is assumed to be rough mess have been removed from the surface of the solid phase. Other chemical, physical as well as mechanical steps may precede the production of the surface of the invention, with the proviso that they do not interfere with the production of the surface.

In The following examples are initially variants of the preparation the surface shown, then Functional tests are described that are low non-specific Detect binding of labeled components to the surface. In the further Examples of the use of the surfaces according to the invention are shown.

Example of a Test procedure.

The regenerated surface is used in contact with a buffer solution containing labeled nucleotides, eg, dUTP-Cy3. After an incubation period of 5 minutes, this solution is removed and the surface is washed with a pure buffer solution. Thereafter, the surface is examined under a fluorescence microscope for the presence of the signals of dUTP-Cy3. An area of approximately 4800 μm ^{2 is} scanned and the fluorescence signals of individual molecules are counted by a program. As a result, the average value representing the density of the signals per 100 μm ^{2 is formed} .

Removal of the outer layer the solid phase, providing a regenerated surface.

Example 1.1:

10 μl of a fresh 1% aqueous HF solution are introduced into the dry microchannel, after 2 min at RT HF solution replaced by a 50 mmol / l Tris-HCl buffer, pH 9.0, and the channel will be five washed with this buffer. The first control of nonspecific Binding of labeled nucleotides and oligonucleotides was carried out immediately after the washing step.

Control of nonspecific binding

The following Tests are used to characterize the non-specific binding:

Test 1.1

10 ul of a 1 .mu.mol / l solution of dCTP-Cy3 (Amersham Bioscience, Freiburg, Germany) in 50 mmol / l Tris-HCl buffer, pH 9.0, is added to the channel and left for 5 min incubated at RT. Connect the channel becomes five sometimes with 50μl 50 mmol / l Tris-HCl buffer, pH 9.0, washed. Signals from the unspecific to the surface bound nucleotides were analyzed under the microscope.

Test 1.2

10 μl of a 1 μmol / l solution of dUTP-AA-SS-Cy3 (synthesized according to instructions in WO 02088382, see 7f-1 and 7f-4 wherein the amino group on the linker is modified with a Cy3 dye, s. Example 2 of the same application) in 50 mmol / l Tris-HCl buffer, pH 9.0, is added to the channel and incubated for 5 min at RT.

Connecting will be the channel five sometimes with 50μl 50 mmol / l Tris-HCl buffer, pH 9.0, washed. Signals from the unspecific to the surface bound nucleotides were analyzed under the microscope.

Test 1.3

10 ul of a 1 .mu.mol / l solution of in each case one labeled oligonucleotide (sequence: see Table 19) in 50 mmol / l Tris-HCl buffer, pH 9.0, is added to the channel and incubated at RT for 5 min. The channel will be connected five times 50μl 50 mmol / l Tris-HCl buffer, pH 9.0. Signals from the unspecific to the surface bound oligonucleotides were analyzed under a microscope.

Test 1.4

10 μl of a 50 μmol / l solution of dUTP-Cy3 (Amersham Bioscience, Freiburg, Germany) in 50th mmol / l Tris-HCl buffer, pH 9.0, is added to the channel and incubated for 5 min at RT. Subsequently, the channel is washed five times with 50 μl 50 mmol / l Tris-HCl buffer, pH 9.0. Signals from the nonspecifically bound to the surface nucleotides were analyzed under the microscope.

Results:

The Data on the binding of labeled substances to the surface are shown in Table 9.

Table 9:

• Treatment of the surface: aqueous HF solution (v / v%)
• Column A) Result of the test 1.1: dCTP-Cy3, 1 μmol / l, 5 'RT, mean value of the measured signals over an area of 100 μm ² ,
• Column B) Result of the test 1.2: dUTP-AA-SS-Cy3, 1 μmol / l, 5 'RT, mean value of the measured signals over an area of 100 μm ² ,
• Column C) Result of the test 1.3: Oligo-T7-19-Cy3, 1 μmol / l, 5 'RT, mean value of the measured signals over an area of 100 μm ² .
• Column D) Result of the test 1.3: Oligo-B1-Cy3, 1 μmol / l, 5 'RT, mean value of the measured signals on an area of 100 μm ² .
• Column E) Result of the test 1.3: Oligo-F1-Cy3, 1 μmol / l, 5 'RT, mean value of the measured signals over an area of 100 μm ² .
• Column F) Result of the test 1.3: Oligo-T40-Cy3, 1 μmol / l, 5 'RT, mean value of the measured signals over an area of 100 μm ² .
• Column G) Result of the test 1.3: oligo-dA26-Cy3, 1 μmol / l, 5 'RT, mean value of the measured signals over an area of 100 μm ² .
• Column H) Result of the test 1.3: Oligo-2-TMR, 1 μmol / l, 5 'RT, mean value of the measured signals over an area of 100 μm ² .
• Column I) Result of the test 1.4: dUTP-Cy3, 50 μmol / l, 5 'RT, mean of the measured signals on an area of 100 μm ² .

The Result shows a very low to low binding of labeled Substances to the surface. The concentrations of labeled nucleotides were 1 μmol / L, since these concentrations in analysis methods as in Example 6.1 and 6.2 be used. The detection device used is for single molecule analysis suitable and is comparable to apparatus for sequencing (WO 03031947). The concentration 50 μmol / l of dUTP-Cy3 shows a low binding of labeled components even at higher Concentrations.

A such density of unspecifically bound signals allows a good Differentiation of individual signals from single molecules at the surface and leads to a lesser overlap with specific signals (see Tables 1 to 8).

The Tests can immediately after an HF treatment or after a certain Time performed become. If the tests should be done after a while, the regenerated surface preferably not dried, but kept in contact with a solution. The test results of Example 1.1 can be saved after one month in 50 mmol / l Tris-HCl, pH 8, RT, with the test 1.1, 1.2, 1.3 and 1.4 are reproduced.

Example 1.2

To illustrate the effect of the removal of the outer layer by the HF treatment results of a surface treatment by a titration series of an HF solution are presented below. The solid phase was treated with different concentrations of the HF solution. The course of the incubation with HF solution and further treatment was similar to that in Example 1.1. In Table 10, results of the test 1.1 and 1.3 with the oligo-T7-19-Cy3 after application are different Concentrations of HF solution shown.

Table 10

• Column A) aqueous HF solution (v / v%)
• Column B) dCTP-Cy3, 1 μmol / l, 5 'RT, mean of the measured signals over an area of 100 μm ² .
• Column C) Oligo-T7-19-Cy3, 1 μmol / l, 5 'RT, mean value of the measured signals over an area of 100 μm ² .

The Results clearly show that the removal of the outer layer the glass phase through the treatment of the solid phase with HF solution to a strong reduction binding of labeled nucleotides and oligonucleotides with an untreated surface.

By the action of the HF solution becomes the outer layer the glass phase removed. The reduction of non-specific binding depends on on the extent of Removal of the outer layer, what in this example by the dependence on the concentration the HF solution becomes clear. The more intense the effect of an HF solution (e.g., by high concentration or elevated temperature or by supplying fresh solution in the channel) the larger the thickness of the removed layer.

successful Removal of the outer layer The solid phase within the meaning of this application was for the following Concentration ranges of the HF solution proved: 0.03 to 0.1 v / v%, 0.1 to 1 v / v%, 1 to 10 v / v%, 4 to 40% by volume.

Preferably For example, the thickness of the ablated layer is one of the following Intervals: from 1 nm to 15 nm, 10 nm to 100 nm, 50 nm to 500 nm, 100 nm to 1000 nm, 0.5 μm up to 5 μm, 1 μm to 20 μm, 5 μm to 100 μm, 20 μm to 500 μm, 100 μm to 2 mm.

Variants of treatment:

Increasing the temperature (eg up to 50 ° C), increasing the time of contact between HF solution and the solid phase (eg up to several hours), adding organic solvent (eg ethanol or DMF up to a concentration of 50%), Addition of other inorganic acids (eg HCl, HNO ₃ or H ₂ SO ₄ ) or a buffering substance such as NH ₄ F does not fundamentally alter the properties of the regenerated surface obtained by the action of the HF solution.

The HF solution can also only after another solution in contact with firmer Be brought phase. For example, the solid phase can only with watery solutions salts (e.g., NaCl), buffers (such as Tris-HCl, borate buffer) or organic solvents (e.g., ethanol, DMF, acetonitrile, acetone) and only then in contact with HF solution to be brought. This is especially true in the production of micro and nano-liquid devices of interest, as different parts of the device are different Need pretreatment can.

Variants of the OF tests

Similar to in test 1.1, 1.2 and 1.4 can also further modifications of nucleotides can be used. It can commercially available Nucleotides with different dyes, such as dUTP-fluorescein, dUTP-TAMRA (NEN, Perkin Elmer Inc.), dUTP-Alexa 555 (Molecular Probes Europe BV, Leiden, The Netherland). Also fissile Nucleotides synthesized as in Example 7 can be used.

The Concentration of the nucleotides is preferably in such tests in the following ranges: 0.1 and 1 μmol / l, 1 and 10 μmol / l, 10 and 100 μmol / l.

Out It follows from the illustrated examples that the HF treatment of the surface is the non-specific binding of nucleotides and oligonucleotides to the surface greatly degrades. This property is essential for analysis methods, which are based on the detection of signals from single molecules.

The solid phase, produced in this way, contains on the surface Si-OH groups, on the further chemical couplings can be made. For example can be nucleic acid chains to the regenerated surface couple.

Example 2.1:

10 μl of a fresh 5 M NaOH solution are introduced into the dry microchannel, after 2 hrs at RT 5 M NaOH solution replaced by a 50 mmol / l Tris-HCl buffer, pH 9.0, and the channel will be five washed with this buffer. The first control of nonspecific Binding of labeled nucleotides and oligonucleotides was carried out immediately after the washing step.

Control of nonspecific Binding:

Test 1.1

10 ul of a 1 .mu.mol / l solution of dCTP-Cy3 (Amersham Bioscience, Freiburg, Germany) in 50 mmol / l Tris-HCl buffer, pH 9.0, is added to the channel and left for 5 min incubated at RT. Connect the channel becomes five times with 50 mmol / l Tris-HCl Buffer, pH 9.0, washed.

Test 1.3

10 ul of a 1 .mu.mol / l solution of Oligo T7-19-Cy3 (Sequence: Table 19) in 50 mmol / l Tris-HCl buffer, pH 9.0, is added to the channel and incubated for 5 min at RT. Connect the channel becomes five times with 50 mmol / l Tris-HCl buffer, pH 9.0, washed.

Results:

The Binding of labeled substances to the surface is shown in Table 11.

Table 11:

• Treatment: 5 mol / l aqueous NaOH solution
• Column A) dCTP-Cy3, 1 μmol / l, 5 'RT, mean of the measured signals over an area of 100 μm ² .
• Column B) Oligo-T7-19-Cy3, 1 μmol / l, 5 'RT, average of the measured signals over an area of 100 μm ² .

The Result shows a very low binding of labeled substances to the surface. The concentrations of labeled nucleotides were 1 μmol / L, since these concentrations were used in analysis procedures as in Example 6.1 become.

Such a density of nonspecifically bound signals allows a good discrimination of individual signals from single molecules at the surface, s. Table 1 to 8 and leads to a lesser over Lappung with specific signals.

Example 2.2

to Vividness of the impact of the removal of the outer layer the NaOH treatment results in a titration series in the following presented. The solid phase was at different concentrations the NaOH solution treated. The titration procedure and further treatment were similar in example 2.1. In Table 12, results of the test are 1.1 and 1.3.

Table 12 Non-specific binding control:

• Column A) aqueous NaOH solution, Indication for minor
• Column B) dCTP-Cy3, 1 μmol / l, 5 'RT, mean of the measured signals over an area of 100 μm ² .
• Column C) Oligo-T7-19-Cy3, 1 μmol / l, 5 'RT, mean value of the measured signals over an area of 100 μm ² .

increase the temperature up to 50 ° C, renewal the time of contact between NaOH solution and the glass phase until to several hours, adding organic solvents such as ethanol or DMF up to concentration of 50%, do not fundamentally change the Properties of the surface, which is caused by the action of the NaOH solution.

By the action of the NaOH solution becomes the outer layer the glass phase removed. The more intense the action of the NaOH solution (e.g., by high concentration or elevated temperature or by Feed fresh solution in the channel) the larger the worn layer.

Preferably the thickness of the abraded layer lies in one of the interwalls of 1 nm and 15 nm, 10 nm and 100 nm, 50 nm and 500 nm, 100 nm and 1000 nm, 0.5 μm and 5 μm, 1 μm and 20 μm, 5 μm and 100 μm, 20 μm and 500 μm, 100 μm and 2 mm.

The NaOH solution can also only after another solution in contact with firmer Be brought phase. For example, the solid phase can only with watery solutions salts (e.g., NaCl), buffers (such as Tris-HCl, borate buffer), or also organic solvents (e.g., ethanol, DMF, acetonitrile) and only then in contact with NaOH solution to be brought. This is especially true in the production of micro and nano-liquid devices of interest, as different parts of the device are different Need pretreatment can.

The in Examples 1 and 2 described methods are suitable for the preparation of solid phases with a surface of low nonspecific Binding of labeled nucleotides.

The solid phase, produced in this way, contains on the surface Si-OH groups, on the further chemical couplings can be made. For example can be nucleic acid chains to the regenerated surface couple.

Example 3: Storage the regenerated solid phase

The regenerated surface of the solid phase obtained in Examples 1 and 2 can be coated with a solution for a few months to have its characteristic of low non-specific binding of labeled substances. maintained. To store the solid phase with a regenerated surface, for example, aqueous salt solutions (eg NaCl up to 1 mol / l), buffer solutions (eg Tris-HCl buffer, pH 7.0 - 9.0, borate buffer, pH 8.0 - 10.0 and many other common buffers such as acetate, phosphate, glycine buffers), aqueous solutions with an organic solvent (such as ethanolic solutions, mixtures of water and DMF or acetonitrile), aqueous solutions with organic additives such as glycerol or glucose, organic solvents such as ethanol, DMF, DMSO, methanol and others. NaN ₃ may be added to prevent bacterial growth.

In a preferred embodiment The invention will provide the solid phase with a regenerated surface throughout subsequent treatment steps not dried out and none Time has a direct contact with air. The storage the freshly regenerated surface takes place in contact with a solution, for example, with water or another solution as described above has been. All other modifying steps in manufacturing done by a solution exchange.

In another embodiment becomes the regenerated surface dried by one of the following procedures:

Procedure A:

• 1) in contact with the surface aqueous Salt or buffer solution is replaced by pure water, all salts by repeated Wash away.
• 2) the water is made by a mixture of one or more organic solvents and water replaced. As organic solvents can one or more water-miscible solvents such as ethanol, methanol, Acetone are used. For example, a mixture of water and ethanol Be used 50:50.
• 3) Main mass of the mixture is removed and the remainder of the mixture be slow from the surface evaporated (e.g., in vacuo).

Procedure B:

• 1) in contact with the surface aqueous Salt or buffer solution is directly through a mixture of one or more organic solvents and water replaced. As organic solvents can one or more water-miscible solvents such as ethanol, methanol, Acetone are used. For example, a mixture of water and ethanol Be used 50:50.
• 2) Main mass of the mixture is removed and the remainder of the mixture be slow from the surface evaporated (e.g., in vacuo).

Procedure C:

• 1) in contact with the surface aqueous Salt or buffer solution is directly by an organic solvent or a mixture from several organic solvents replaced. As organic solvents can one or more water-miscible solvents such as ethanol, methanol, acetone be used.
• 2) Main mass of the mixture is removed and the remainder of the mixture be slow from the surface evaporated (e.g., in vacuo).

The freshly regenerated surfaces, which have undergone a drying step, have higher nonspecific Binding of labeled nucleotides to, as the surfaces without Drying step.

Out For this reason, all subsequent treatment steps are only for surfaces of the described after the step of erosion of the solid phase outer layer do not go through a drying step.

Examples of fixation of nucleic acids

Example 4.

modification the regenerated surface with a reactive linker

Modification of the regenerated surfaces obtained under Example 1, 2 or 3 takes place, for example with silylation reagents. Many examples of silica modification are known ("Silane Coupling Agents" 2 Ed. 1991, ISBNO-306-43473-3).

Example 4.1 Modification the surface with glycidyloxypropyl trimethoxysilane

regenerated surface the solid phase in this example forms part of a Microfluidic channel represents.

• 1. Puffer-Lösung im Mikroflüssigkeitskanal wurde gegen reines Wasser (Biddest-Qualität) ausgetauscht: Der Mikrokanal mit der regenerierten Oberfläche der festen Phase wurde mit 200 μl Wasser gespült.
• 2. Das Wasser wurde gegen Aceton, 100%, ausgetauscht, indem der Mikrokanal mit 500 μl Aceton gewaschen wurde.
• 3. Glycidyloxypropyl-Trimethoxisilan, 100%, wurde in den Mikrokanal eingeführt, bis Aceton komplett ausgetauscht wurde (100 μl). Die Reaktion dauerte 1 h, bei RT.
• 4. Glycidyloxypropyl-Trimethoxisilan wurde durch Aceton, 100%, ausgetauscht, 1 ml.
• 5. Aceton wurde durch Wasser, 200 μl, ersetzt.

The regenerated solid phase was obtained as in Example 1.1 and processed further without drying out as follows:

• 1. Buffer solution in the microfluidic channel was replaced with pure water (Biddest grade): The microchannel with the regenerated surface of the solid phase was rinsed with 200 μl of water.
• 2. The water was replaced with acetone, 100%, by washing the microchannel with 500 μl of acetone.
• 3. Glycidyloxypropyl trimethoxysilane, 100%, was introduced into the microchannel until acetone was completely replaced (100 μl). The reaction lasted 1 h, at RT.
• 4. Glycidyloxypropyl trimethoxysilane was replaced by acetone, 100%, 1 ml.
• 5. Acetone was replaced by water, 200 μl.

On this way, regenerated surface was activated with epoxy groups. To this surface can now different substances are coupled.

acetone For example, by another organic solvent be replaced, for example, ethanol, DMAA, DMSO or DMF.

The Extent of Modification of the surface can by the time, concentration of reagents and temperature to be influenced. Instead of 100% glycidyloxypropyl trimethoxysilane can also another Silylisrungsreagez or a mixture of several Silylation reagents are used. It can be different functional groups are coupled to the surface. silylating can in organic solvents, diluted as DMAA, DMF, DMSO become. It can also watery Mixtures are used. The time of the modification may e.g. between a few minutes and several hours.

By Silylierungsreagentien can different reactive groups, for example epoxies, - carboxy-, Acrylic, mercapto, aldehyde groups are coupled to the surface. The vote a corresponding group depends on compatibility with reagents, in later Steps come in contact with surface.

By the introduction a linker component with an active group can have more Modifications are made. For example, more Linker can be connected.

In this example became the surface after coupling the linker component not dried out, but in a buffer solution stored until the coupling of other substances.

The performance of tests 1.1, 1.2, 1.3, 1.4 showed that the non-specific binding of test substances to the surface was very low and the mean value of the density of the signals was below 10 per 100 μm ² .

Worse test results were obtained by modifying the surfaces by introducing amino groups. Without being bound to any particular theory, it is believed that the amino-modified surfaces are positively charged in aqueous buffer solutions (especially at pH values around 9), resulting in enhanced non-specific binding of nucleic acid chains and nucleotides to the surface The result of test 1.1 (see example 1.1) gave more than 100 signals per 100 μm ² .

Example 5

Specific coupling of nucleic acids to the surface.

The binding of nucleic acids to the surface may, for example, be at one of the two ends of the chain or via a group coupled to one of the nucleotides within the chain. The covalent coupling of nucleic acids can be linked to the surface-bound linker or directly to the glass surface. The affine binding can be effected, for example, between a streptavidin fixed on the surface and the biotin bound to the nucleic acid. Another example of the affinity binding of nucleic acids is the hybridization of complementary nucleic acid chains to the nucleic acids fixed on the surface. Examples of the covalent and affine coupling of the nucleic acid chains to the surface are shown below.

Example 5.1 covalent coupling

5.1.1 Covalent coupling of nucleic acid chains to the active groups of a linker on the surface

In In this example, the nucleic acid chains contained one at the 3 'position Left (6-atom) coupled amino group and at 5'-position one via a linker coupled Fluorescent dye. The amino group can be attached to the Epoxy group of the linker component of the solid phase can be coupled. The coupling of the nucleic acid strands to the surface was detected by the Detection of the to the nucleic acid coupled dye.

The regenerated and epoxy-activated surface obtained as in Example 4.1 was processed further directly:
Water was exchanged with 10 μl of 50 mmol / l borate buffer pH 10.0 and immediately afterwards 10 μl of a 30 μmol / l solution of Cy3-oligo-dT31-NH ₂ (sequence: Table 18) in 50 mmol / l borate buffer , pH 10, introduced into the channel. The reaction lasted 12 h at RT. Thereafter, the microchannel was washed with 100 μl of 50 mmol / l borate buffer, pH 9.0.

The resulting surface of the solid phase was checked for the presence of signals from labeled nucleic acids. The result showed that the nucleic acid strands were coupled to the surface at a density of 40 to 200 strands / 100 μm ² . By varying the concentration of the oligonucleotides with amino groups and / or the time and / or temperature of the incubation, it was also possible to bind nucleic acid strands to the surface in other densities. The following ranges of densities of nucleic acid strands per 100 μm ^{2 were} achieved: 10 to 50, 50 to 100, 100 to 200, 200 to 1000, 1000 to 5000.

5.1.2 Covalent coupling of nucleic acid chains to the active groups of a linker on the surface

In In this example, the nucleic acid chains contained one at the 3 'position Left (6-atom) coupled amino group. The 5 'position of the nucleic acid chain contained no fluorescent dye. The amino group can be attached to the Epoxy group of the linker component of the solid phase can be coupled. Coupling was detected by hybridization of a complementary oligonucleotide, which was labeled with a fluorescent dye. The detection took place by detecting the signals from the hybridized nucleic acid strands.

The regenerated and epoxy-activated surface obtained as in Example 4.1 was processed further directly:
Water was exchanged with 10 μl of 50 mmol / l borate buffer pH 10.0 and immediately afterwards 10 μl of a 30 μmol / l solution of oligo-dT31-NH ₂ (sequence see Table 18) in 50 mmol / l borate buffer, pH 10, introduced into the channel. The reaction lasted 12 h at RT. Thereafter, the microchannel was washed with 100 μl of 50 mmol / l borate buffer, pH 9.0.

On this way became a surface obtained carrying covalently fixed nucleic acid chains. The surface became not dried out. She could continue to be used directly or in a buffer solution e.g. Store phosphate buffer 50 mmol / l pH 8 until the next steps were made.

Testing the surface:

Testing the nonspecific Binding to the solid phase

Examination of the non-specific binding of the labeled nucleotides and oligonucleotides to the surface obtained (Test 1.1, 1.2, 1.3, 1.4) showed a very low unspecific binding to the surface. The density of the signals due to non-specific binding was less than 10 per 100 μm ² .

Table 13

• Column A) Result of the test 1.1: dCTP-Cy3, 1 μmol / l, 5 'RT, mean value of the measured signals over an area of 100 μm ² ,
• Column B) Result of the test 1.2: dUTP-AA-SS-Cy3, 1 μmol / l, 5 'RT, mean value of the measured signals over an area of 100 μm ² ,
• Column C) Result of the test 1.3 ( 7A ): Oligo-T7-19-Cy3, 1 μmol / l, 5 'RT, average of the measured signals over an area of 100μm ² .
• Column D) Result of the test 1.4: dUTP-Cy3, 50 μmol / l, 5 'RT, mean value of the measured signals over an area of 100 μm ² .

Testing the hybridization a complementary one oligonucleotide:

The surface obtained here the solid phase was on presence the coupled nucleic acid chains checked. For that were 10 μl of a 100 nmol / l solution with oligo-dA26-Cy3 (Sequence see Table 18) in 50 mmol / l Tris-HCl, pH 9.0, for 5 min at RT in contact with the surface brought. Subsequently became the surface washed with 50 mmol / l Tris-HCl pH 9.0 and it was detected the signals of hybridized nucleic acids.

The result showed that the nucleic acid strands were coupled to the surface at a density of 100 to 200 strands / 100 μm ² ( 7B ).

By varying the concentration of the oligonucleotides with amino groups and / or the time and / or temperature of the incubation in the covalent coupling succeeded nucleic acid strands in other density to the surface to bind. The following ranges of densities of nucleic acid strands per 100 μm ^{2 were} achieved: 10 to 50, 50 to 200, 100 to 400, 200 to 1000, 1000 to 5000.

5.1.3 Covalent coupling of nucleic acid chains to the active groups of a linker on the surface

In In this example, the nucleic acid chains contained one at the 5 'position Left (6-atom) coupled amino group. The 3 'position of the nucleic acid chain was free. The amino group can be attached to the epoxide group of the linker component of the solid Phase are coupled. These nucleic acid chains can be used as primers. Coupling was detected by hybridization of a complementary oligonucleotide, which was labeled with a fluorescent dye. The detection took place by detecting the signals from the hybridized nucleic acid strands.

The regenerated and epoxy-activated surface obtained as in Example 4.1 was processed further directly:
Water was exchanged with 10 μl of 50 mmol / l borate buffer pH 10.0 and immediately afterwards 10 μl of a 30 μmol / l solution of oligo-dA50-NH ₂ (sequence see Table 18) in 50 mmol / l borate buffer, pH 10, introduced into the channel. The reaction lasted 12 h at RT. Thereafter, the microchannel was washed with 100 μl of 50 mmol / l borate buffer, pH 9.0.

On this way became a surface obtained carrying covalently fixed nucleic acid chains. The surface became not dried out. She could continue to be used directly or in a buffer solution e.g. Store phosphate buffer 50 mmol / l pH 8 until the next steps were made.

Testing the surface:

Testing the nonspecific Binding to the solid phase

Examination of the non-specific binding of the labeled nucleotides and oligonucleotides to the surface obtained (Test 1.1, 1.2, 1.3, 1.4) showed a very low unspecific binding to the surface. The density of the signals due to non-specific binding was less than 10 per 100 μm ² .

Testing the hybridization a complementary one oligonucleotide:

The resulting surface the solid phase was on presence the coupled nucleic acid chains checked. For that were 10 μl of a 100 nmol / l solution with oligo-dT30-Cy3 (see Table 18) 100 nmol / l in 50 mmol / l Tris-HCl, pH 9.0, for Brought into contact with the surface at RT for 5 min. Subsequently, the surface washed with 50 mmol / l Tris-HCl pH 9.0 and it was detected the signals of hybridized nucleic acids.

The result showed that the nucleic acid strands were coupled to the surface at a density of 100 to 400 strands / 100 μm ² .

By varying the concentration of the oligonucleotides with amino groups and / or the time of incubation in the covalent coupling succeeded nucleic acid strands in other densities to the surface to bind. The following ranges of densities of nucleic acid strands per 100 μm ^{2 were} achieved: 10 to 50, 50 to 100, 100 to 400, 200 to 1000, 1000 to 5000.

5.1.4 direct covalent Coupling of nucleic acid chains to the surface

In In this example, the nucleic acid chains contained one at the 3 'position Left-coupled trimethoxysilyl group. Such oligonucleotides can from Thermo Electron Corporation GmbH, Ulm, Germany, be obtained. The 5'-position the nucleic acid chain contained no fluorescent dye. Proof of coupling took place over Hybridization of a complementary Oligonucleotide labeled with a fluorescent dye. Detection was by detection of the signals from the hybridized Nucleic acid strands.

• 1. Puffer-Lösung im Mikroflüssigkeitskanal wurde gegen reines Wasser (Biddest-Qualität) ausgetauscht: Der Mikrokanal mit der regenerierten Oberfläche der festen Phase wurde mit 200 μl Wasser gespült.
• 2. Das Wasser wurde gegen Aceton, 100%, ausgetauscht, indem der Mikrokanal mit 500 μl Aceton gewaschen wurde.
• 3. 10 μl einer 0,5 μmol/l Lösung von Oligo-dT31-Si(O-Me)₃ (Sequenz s. Tabelle 18) in DMF wurden in den Mikrokanal gegeben. Die Reaktion verlief 30 min bei RT.
• 4. Der Mikrokanal wurde mit Aceton 100% 1ml gewaschen.
• 5. Aceton wurde durch Wasser (Biddest-Qualität) ersetzt (200 μl)
• 6. Eine Puffer-Lösung (50 mmol/l Phosphat-Puffer, pH 8,0) wurde in den Mikrokanal gegeben. In dieser Lösung wurde die feste Phase aufbewahrt.

The surface obtained as in Example 1.1 was further processed directly. The following steps were carried out:

• 1. Buffer solution in the microfluidic channel was replaced with pure water (Biddest grade): The microchannel with the regenerated surface of the solid phase was rinsed with 200 μl of water.
• 2. The water was replaced with acetone, 100%, by washing the microchannel with 500 μl of acetone.
• 3. 10 μl of a 0.5 μmol / l solution of oligo-dT31-Si (O-Me) ₃ (sequence see Table 18) in DMF were added to the microchannel. The reaction proceeded for 30 min at RT.
• 4. The microchannel was washed with acetone 100% 1ml.
• 5. Acetone was replaced by water (Biddest quality) (200 μl)
• 6. A buffer solution (50 mmol / L phosphate buffer, pH 8.0) was added to the microchannel. In this solution, the solid phase was stored.

On this way became a surface obtained carrying covalently fixed nucleic acid chains. The surface of the solid phase was not dried out. She was able to continue directly used or in a buffer solution e.g. Store phosphate buffer 50 mmol / l pH 8 until the next steps were made.

acetone For example, by another organic solvent be replaced, for example DMAA, DMSO or DMF or acetonitrile.

The Extent of Modification of the surface can by the time, concentration of reagents and temperature to be influenced. Instead of a trimethoxysilyl group can also another silylation reaction coupled to the oligonucleotide be used. Oligonucleotides with activated silyl groups can in organic solvents, such as DMAA, DMF, DMSO or acetonitrile. The time of the modification may e.g. between a few minutes and several hours. The choice of a corresponding active silyl group depends on compatibility with reagents that in later Steps in contact with the surface come.

Testing the surface:

Testing the nonspecific Binding to the solid phase

Examination of the non-specific binding of the labeled nucleotides and oligonucleotides to the surface obtained (Test 1.1, 1.2, 1.3, 1.4) showed a very low unspecific binding to the surface. The density of the signals due to non-specific binding was less than 10 per 100 μm ² .

Table 14

• Column A) Result of the test 1.1: dCTP-Cy3, 1 μmol / l, 5 'RT, mean value of the measured signals over an area of 100 μm ² ,
• Column B) Result of the test 1.2: dUTP-AA-SS-Cy3, 1 μmol / l, 5 'RT, mean value of the measured signals over an area of 100 μm ² ,
• Column C) Result of the test 1.3 ( 8A ): Oligo-T7-19-Cy3, 1 μmol / l, 5 'RT, average of the measured signals over an area of 100μm ² .
• Column D) Result of the test 1.4: dUTP-Cy3, 50 μmol / l, 5 'RT, mean value of the measured signals over an area of 100 μm ² .

Testing the hybridization a complementary one Oligonucleotides:

The resulting surface the solid phase was on presence the coupled nucleic acid chains checked. For that were 10 μl of a 100 nmol / l solution with oligo-dA26-Cy3 (sequence see Table 18) in 50 mmol / l Tris-HCl, pH 9.0, for Brought into contact with the surface at RT for 5 min. Subsequently was the surface washed with 50 mmol / l Tris-HCl pH 9.0 and it was detected the signals of hybridized nucleic acids.

The result showed that the nucleic acid strands were coupled to the surface at a density between 300 and 1000 strands / 100 μm ² ( 8B ).

By varying the concentration of the oligonucleotides with reactive silyl groups and / or the time of incubation in the covalent coupling succeeded nucleic acid strands in other densities to the surface to bind. The following ranges of densities of nucleic acid strands per 100 μm ^{2 were} achieved: 10 to 50, 50 to 100, 100 to 400, 200 to 1000, 1000 to 5000.

5.1.5 direct covalent Coupling of nucleic acid chains to the surface

In In this example, the nucleic acid chains contained one at the 5 'position Left-coupled trimethoxysilyl group. The 3 'position of the nucleic acid chain is free, so that the nucleic acid chain could serve as a primer. Coupling was detected by hybridization a complementary one Oligonucleotide labeled with a fluorescent dye. Detection was by detection of the signals from the hybridized Nucleic acid strands.

• 1. Puffer-Lösung im Mikroflüssigkeitskanal wurde gegen reines Wasser (Biddest-Qualität) ausgetauscht: Der Mikrokanal mit der regenerierten Oberfläche der festen Phase wurde mit 200 μl Wasser gespült.
• 2. Das Wasser wurde gegen Aceton, 100%, ausgetauscht, indem der Mikrokanal mit 500 μl Aceton gewaschen wurde.
• 3. 10 μl einer Lösung von Oligo-dA50-Si(O-Me)₃ (Sequenz s. Tabelle 18) 0,5 μmol/l in DMF wurden in den Mikrokanal gegeben. Die Reaktion verlief 30 min bei RT.
• 4. Der Mikrokanal wurde mit Aceton 100% 1ml gewaschen.
• 5. Aceton wurde durch Wasser (Biddest-Qualität) ersetzt (200 μl)
• 6. Eine Puffer-Lösung (50 mmol/l Phosphat-Puffer, pH 8,0) wurde in den Mikrokanal gegeben. In dieser Lösung wurde die feste Phase aufbewahrt.

The surface obtained in Example 1.1 was further processed directly. The following steps were carried out:

• 1. Buffer solution in the microfluidic channel was replaced with pure water (Biddest grade): The microchannel with the regenerated surface of the solid phase was rinsed with 200 μl of water.
• 2. The water was replaced with acetone, 100%, by washing the microchannel with 500 μl of acetone.
• 3. 10 μl of a solution of oligo-dA50-Si (O-Me) ₃ (sequence see Table 18) 0.5 μmol / l in DMF was added to the microchannel. The reaction proceeded for 30 min at RT.
• 4. The microchannel was washed with acetone 100% 1ml.
• 5. Acetone was replaced by water (Biddest quality) (200 μl)
• 6. A buffer solution (50 mmol / L phosphate buffer, pH 8.0) was added to the microchannel. In this solution, the solid phase was stored.

On this way became a surface obtained carrying covalently fixed nucleic acid chains.

The surface the solid phase was not dried out. She was able to continue directly used or in a buffer solution e.g. Store phosphate buffer 50 mmol / l pH 8 until the next steps were made.

acetone For example, by another organic solvent be replaced, for example DMAA, DMSO or DMF or acetonitrile.

The Extent of Modification of the surface can by the time, concentration of reagents and temperature to be influenced. Instead of a trimethoxysilyl group can also another silylation reaction coupled to the oligonucleotide be used. Oligonucleotides with activated silyl groups can in organic solvents, such as DMAA, DMF, DMSO or acetonitrile. It can also watery Mixtures are used. The time of the modification may e.g. between a few minutes and several hours. The choice of a corresponding active silyl group depends on compatibility with reagents, in later Steps in contact with the surface come.

Testing the surface:

Testing the nonspecific Binding to the solid phase:

Examination of the non-specific binding of the labeled nucleotides and oligonucleotides to the surface obtained (Test 1.1, 1.2, 1.3, 1.4) showed a very low unspecific binding to the surface. The density of the signals was less than 10 per 100 μm ² .

Testing the hybridization a complementary one Oligonucleotides:

The resulting surface the solid phase was on presence the coupled nucleic acid chains checked. For that were 10 μl of a solution with oligo-dT30-Cy3 (sequence see Table 18) 1 nmol / l in 50 mmol / l Tris-HCl, pH 9.0, for Brought into contact with the surface at RT for 5 min. Subsequently was the surface washed with 50 mmol / l Tris-HCl pH 9.0 and it was detected the signals of hybridized nucleic acids.

The result showed that the nucleic acid strands were coupled to the surface at a density of 200 to 1000 strands / 100 μm ² .

By varying the concentration of the oligonucleotides with reactive silyl groups and / or the time and / or the temperature of the incubation in the covalent coupling succeeded nucleic acid strands in other densities to the surface to bind. The following ranges of densities of nucleic acid strands per 100 μm ^{2 were} achieved: 10 to 50, 50 to 100, 100 to 200, 200 to 1000, 1000 to 5000.

5.2 Affine binding of nucleic acid chains to the surface:

5.2.1 binding to the the surface fixed streptavidin

In this example was first Streptavidin to the surface coupled. Subsequently became the nucleic acid chains to the streptavidin over Streptavidin-biotin group bound. The nucleic acid chains contributed at the 5'-position one over a linker (TEG) coupled biotin group. The 3 'position of the nucleic acid chain was free. These nucleic acid chains can be used as a primer. Coupling was detected by hybridization a complementary one Oligonucleotide labeled with a fluorescent dye. Detection was by detection of the signals from the hybridized Nucleic acid strands.

The regenerated and epoxy-activated surface obtained as in Example 4.1 was processed further directly:
Water was exchanged with 10 μl of 50 mmol / l borate buffer pH 10.0 and immediately thereafter 10 μl of a 1 μg / μl solution of streptavidin in 50 mmol / l borate buffer, pH 10, were introduced into the channel. The reaction lasted 2 h at RT. Thereafter, the microchannel was washed with 100 μl of 50 mmol / l borate buffer, pH 9.0. Subsequently, 10 μl of a 1 μmol / l solution of dT40-biotin (sequence see Table 18) in Tris-HCl were introduced into the microchannel and incubated for 5 min at RT. Thereafter, the microchannel was washed with 200 μl of 50 mmol / l Tris-HCl buffer, pH 9.0.

On this way became a surface obtained affinely affixed nucleic acid chains. The surface was not dried out. She could continue to be used directly or in a buffer solution e.g. Store phosphate buffer 50 mmol / l pH 8 until the next steps were made.

Testing the surface:

Testing the nonspecific Binding to the solid phase:

Examination of the non-specific binding of the labeled nucleotides and oligonucleotides to the surface obtained (Test 1.1, 1.3, 1.4) showed a very low unspecific binding to the surface. The density of the signals was less than 10 per 100 μm ² .

Testing the hybridization a complementary one Oligonukieotides:

The resulting surface the solid phase was on presence the coupled nucleic acid chains checked. For that were 10 μl of a 100 nmol / l solution with oligo-dA26-Cy3 (sequence see Table 18) in 50 mmol / l Tris-HCl, pH 9.0, for Brought into contact with the surface at RT for 5 min. Subsequently was the surface washed with 50 mmol / l Tris-HCl pH 9.0 and it was detected the signals of hybridized nucleic acids.

The result showed that the nucleic acid strands were coupled to the surface at a density of 200 to 1000 strands / 100 μm ² .

By varying the concentration or time of the incubation of oligonucleotides with biotin groups and / or the concentration and / or time of incubation in the covalent coupling of streptavidin succeeded nucleic acid strands to bind to the surface in other densities. The following ranges of densities of nucleic acid strands per 100 μm ^{2 were} achieved: 10 to 50, 50 to 100, 100 to 200, 200 to 1000, 1000 to 5000.

Example 6

• a) zu den an die Oberfläche gebundenen NSKF-Primer-Komplexen eine Lösung zugibt, die eine oder mehrere Polymerasen und ein bis vier modifizierte Nukleotide (NTs*) enthält, die mit Fluoreszenzfarbstoffen markiert sind, wobei die bei gleichzeitiger Verwendung von mindestens zwei NTs* jeweils an den NTs* befindlichen Fluoreszenzfarbstoffe so gewählt sind, dass sich die verwendeten NTs* durch Messung unterschiedlicher Fluoreszenzsignale voneinander unterscheiden lassen, wobei die NTs strukturell an der Base so modifiziert sind, dass die Polymerase nach Einbau eines solchen NT* in einen wachsenden komplementären Strang nicht in der Lage ist, ein weiteres NT* in denselben Strang einzubauen, wobei der Fluoreszenzfarbstoff abspaltbar ist und die strukturelle Modifikation ein abspaltbarer sterisch anspruchsvoller Ligand ist, man
• b) die in Stufe a) erhaltene stationäre Phase unter Bedingungen inkubiert, die zur Verlängerung der komplementären Stränge geeignet sind, wobei die komplementären Stränge jeweils um ein NT* verlängert werden, man
• c) die in Stufe b) erhaltene stationäre Phase unter Bedingungen wäscht, die zur Entfernung nicht in einen komplementären Strang eingebauter NTs* geeignet sind, man
• d) die einzelnen, in komplementäre Stränge eingebauten NTs* durch Messen des für den jeweiligen Fluoreszenzfarbstoff charakteristischen Signals detektiert, wobei man gleichzeitig die relative Position der einzelnen Fluoreszenzsignale auf der Reaktionsoberfläche bestimmt, man
• e) zur Erzeugung unmarkierter (NTs oder) NSKFs die Fluoreszenzfarbstoffe und die sterisch anspruchsvollen Liganden von den am komplementären Strang angefügten NTs* abspaltet, man
• f) die in Stufe e) erhaltene stationäre Phase unter Bedingungen wäscht, die zur Entfernung der Fluoreszenzfarbstoffe und der Liganden geeignet sind, man die Stufen a) bis f) gegebenenfalls mehrfach wiederholt,

wobei man die relative Position einzelner NSKF-Primer-Komplexe auf der Reaktionsoberfläche und die Sequenz dieser NSKFs durch spezifische Zuordnung der in Stufe d) in aufeinanderfolgenden Zyklen an den jeweiligen Positionen detektierten Fluoreszenzsignale zu den NTs bestimmt.Here is an example of a method is shown in which the surface of the invention can be used. A highly parallel sequencing method of single nucleic acid chain molecules, in which one
Fragments (NSKFs) of single-stranded NSKs of about 50 to 1000 nucleotides in length, representing overlapping partial sequences of the total sequences;
bind the NSKFs in a random array using a single or multiple different primers in the form of NSKF primer complexes on a reaction surface;
Perform a cyclic assembly reaction of the complementary strand of NSKFs using one or more polymerases by

• a) adding to the surface-bound NSKF-primer complexes a solution containing one or more polymerases and one to four modified nucleotides (NTs *) labeled with fluorescent dyes which, when simultaneously using at least two NTs * each of the NTs * located fluorescent dyes are selected so that the NTs used * differ by measuring different fluorescence signals from each other, the NTs are structurally modified at the base so that the polymerase after incorporation of such NT * in a growing complementary strand is unable to incorporate another NT * in the same strand, wherein the fluorescent dye is cleavable and the structural modification is a cleavable sterically demanding ligand, one
• b) the stationary phase obtained in step a) is incubated under conditions suitable for extending the complementary strands, the complementary strands being extended by one NT * each;
• c) washing the stationary phase obtained in stage b) under conditions which are suitable for the removal of non-complementary NTs *
• d) the individual NTs * incorporated in complementary strands are detected by measuring the signal characteristic of the respective fluorescent dye, at the same time determining the relative position of the individual fluorescence signals on the reaction surface;
• e) for the production of unlabeled (NTs or) NSKFs, the fluorescent dyes and the sterically demanding ligands of the NTs attached to the complementary strand * splits, one
• f) washing the stationary phase obtained in step e) under conditions which are suitable for removing the fluorescent dyes and the ligands, if necessary repeating steps a) to f) several times,

the relative position of individual NSKF primer complexes on the reaction surface and Se The sequence of these NSKFs is determined by specific assignment of the fluorescence signals to the NTs detected in step d) in successive cycles at the respective positions.

Out The determined partial sequences can be, for example, the overall sequence determine the NSKs. Under a parallel sequence analysis is in In this context, the simultaneous sequence analysis of many NSKFs understood (for example, 1,000,000 to 10,000,000), these being NSKFs from a single NSK population or from several different ones NSK populations are derived.

The preserved population of overlapping Partial sequences can be for example, in de novo sequencing with commercially available Assemble programs to the overall sequence of the NSK (Huang et al., Genom Res. 1999 v.9 p.868, Huang Genomics 1996 v.33 p.21, Bonfield et al. NAR 1995, v.23 p.4992, Miller et al. J.Comput.Biol. 1994 v.1 p.257).

at the analysis of variants of a known reference sequence mutations or single nucleotide polymorphisms by comparison the obtained overlapping Detect partial sequences with the reference sequence.

• a) in jedem Zyklus nur jeweils ein markiertes NT*,
• b) in jedem Zyklus jeweils zwei unterschiedlich markierte NTs* oder
• c) in jedem Zyklus jeweils vier unterschiedlich markierte NTs*

einsetzt.According to a particular embodiment of the invention, the process can be carried out by repeatedly repeating steps a) to f) of the cyclic synthesis reaction, wherein

• a) in each cycle only one marked NT *,
• b) in each cycle two different marked NTs * or
• c) four differently marked NTs in each cycle *

starts.

If the NSKs variants of a known reference sequence are the Procedure also performed be, by the stages a) to f) of the cyclic synthesis reaction repeated several times, alternating in each cycle use two differently marked NTs * and two unmarked NTs and the total sequences by comparison with the reference sequence determined.

The inventive method serves for the determination of the nucleic acid sequences and can in different Fields of genetics are used. These include in particular the determination unknown, long sequences, analyzes of sequence polymorphisms and Point mutations as well as the parallel analysis of a large number on gene sequences.

The Preparation of the material to be analyzed (single- and double-stranded nucleic acid sequences) depends on the task and has the goal of a long nucleic acid chain a population of relatively small, single-stranded nucleic acid chain fragments (NSKFs) to form these fragments with one for the start of the sequencing reaction suitable primer (NSKF primer complexes) and to fix on a flat surface.

there Individual NSKFs will be on a flat surface in such a way fixed that an enzymatic reaction take place on these molecules can. In principle, different types of immobilization are possible the purpose, the nature of the NSK and the response used Depend on polymerase. The NSKFs become random when immobilized or bound the surface distributed, i. it must be so not paid attention to an exact positioning of the individual chains become. NACF primer complexes can over the NSKFs or primers to the surface be bound. The NSKF primer complexes must be in such a Density on the surface be fixed, that a clear assignment of the later detected Ensures signals from the built-in NT * s to individual NSKFs is.

To The preparation of the NSKFs starts with all surfaces immobilized on the surface NACF primer complex molecules the sequencing reaction. As a basis of sequencing serves the synthesis of the complementary Stranges to each individual bound NSKF. It will be in the newly synthesized strand labeled NTs * incorporated. The polymerase builds only a single tagged NT * into the growing chain. This is due to the reversible coupling one for termination leading, sterically demanding group to the NTs * achieved. The installation another marked NT * is thereby made impossible. This steric Sophisticated group is preferably a fluorescent dye.

• a) Zugabe einer Lösung mit markierten Nukleotiden (NTs*) und Polymerase zu den gebundenen NSKF-Primer-Komplexen,
• b) Inkubation der gebundenen NSKF-Primer-Komplexe mit dieser Lösung unter Bedingungen, die zur Verlängerung der komplementären Stränge um ein NT geeignet sind,
• c) Waschen,
• d) Detektion der Signale von einzelnen Molekülen,
• e) Entfernung der Markierung von den eingebauten Nukleotiden,
• f) Waschen.

The sequencing reaction proceeds in several cycles. A cycle includes the following steps:

• a) Addition of a solution with labeled nucleotides (NTs *) and polymerase to the bound NSKF Pri mer complexes
• b) incubation of the bound NSKF primer complexes with this solution under conditions suitable for extension of the complementary strands around an NT,
• c) washing,
• d) detection of the signals of individual molecules,
• e) removal of the label from the incorporated nucleotides,
• f) washing.

Possibly a multiple repetition of the cycle (a-f) takes place.

The Reaction conditions of step (b) in one cycle become so selected that the polymerases on more than 50% of the sequencing reaction involved NSKFs (Extensible NSKF primer complexes) in one cycle, insert a labeled NT * can, preferably more than 80%.

The Number of performed Cycles depends thereby from the respective task off, is theoretically not limited and is preferably between 10 and 200.

After that is for each pinned NSKF has its specific sequence out of order the built-in NTs * determined.

Out the overlapping one NSKF sequences may in one embodiment be the original NSK sequence can be reconstructed ("Automated DNA sequencing and analysis" p. 231 et seq. 1994 M. Adams et al. Academic Press, Huang et al. Genome Res. 1999 v.9 P.868, Huang Genomics 1996 v.33 p.21, Bonfield et al. NAR 1995 v.23 P.4992, Miller et al. J.Comput.Biol. 1994 v.1 p.257). Looking for it in the entire population of NSKF sequences for matches / overlaps in the sequences of NSKFs.

there can The errors of the method are detected and corrected by various means become. All Steps of the procedure can be largely automated.

• 1. Da die Moleküle einzeln detektiert werden, besteht keine Gefahr, dass das Signal durch die Desynchronisation in der Population fehlerhaft wird. Für jedes fixierte NSKF wird eine eigene Sequenz erstellt. Daher spielt es keine Rolle, ob an einem benachbarten Molekül die Synthese bereits weiter fortgeschritten oder zurückgeblieben ist.
• 2. Es ist nicht notwendig, Moleküle in einer definierten Anordnung auf der Oberfläche zu fixieren, da das Signal von einzelnen Molekülen ausgeht und nicht von einer räumlich definierten Population (was bei BASS-Methoden notwendig ist).

Working with single molecules has several advantages over the previously described BASS method:

• 1. Since the molecules are detected individually, there is no danger that the signal will be corrupted by the desynchronization in the population. For each fixed NSKF a separate sequence is created. Therefore, it does not matter if the synthesis of an adjacent molecule is more advanced or lagging behind.
• 2. It is not necessary to fix molecules in a defined arrangement on the surface, since the signal emanates from single molecules and not from a spatially defined population (which is necessary in BASS methods).

Selection of the material

With Help the method of the invention Is it possible, both preselected DNA sequences (e.g. in YAC, PAC, or BAC vectors (R. Anand et al., NAR 1989 v.17 P.3425, H. Shizuya et al. PNAS 1992 v.89 p.8794, "Construction of bacterial artificial chromosomal libraries using the modified PAC system "in" Current Protocols in Human Genetics "1996 John Wiley & Sons Inc.) cloned portions of a genome) as well as not preselected To analyze DNA (e.g., genomic DNA, cDNA mixtures). By a Preselection it is possible beforehand relevant information, e.g. Sequence sections from a genome or populations of gene products, from the large amount to filter out genetic information and thus the amount of restrict sequences to be analyzed.

preparation of the material

aim Material preparation is bound single-stranded NSKFs with a length preferably 50-1000 NTs, a single primer binding site and a hybridized one To obtain primers (bound NSKF primer complexes). In detail can very variable constructions derived from this general structure become. To improve the clarity, here are some examples the cited Methods can be used individually or in combination.

Generation of short nucleic acid chain fragments (50-1000 NTs) (fragmentation step)

Important is that the fragmentation of NSKs occurs in such a way that fragments to be obtained, the overlapping Represent partial sequences of the overall sequences. This is done by procedures reached, in which fragments of different lengths as fission products in random distribution arise.

According to the invention, the Generation of nucleic acid chain fragments (NSKFs) by several methods, e.g. through the fragmentation the starting material with ultrasound or by endonucleases ("Molecular cloning" 1989 J. Sambrook et al. Cold Spring Harbor Laboratory Press), e.g. by unspecific Endonukleasegemische. According to the invention, the ultrasound fragmentation prefers. You can adjust the conditions so that fragments with an average length from 100 bp to 1 kb. These fragments are subsequently attached their ends by the Klenow fragment (E. coli polymerase I) or filled by the T4 DNA polymerase ("Molecular cloning" 1989, J. Sambrook et al., Cold Spring Harbor Laboratory Press).

Moreover can from a long NSK using randomized primer complementary short NSKFs are synthesized. This method is particularly preferred in the analysis of gene sequences. In this case, single-stranded DNA fragments on the mRNA formed with randomized primers and a reverse transcriptase (Zhang-J et al., Biochem., 1999, v.337, p.231, Ledbetter et al., J. Biochem., 1994 v.269 p.31544, Kolls et al. Anal. 1993 v.208 p.264, decraene et al. Biotechniques 1999 v.27 S.962).

Introduction of a Primary binding site in the NSKF.

The Primer binding site (PBS) is a sequence segment that is a selective To allow binding of the primer to the NSKF.

In an embodiment can the primer binding sites will be different so that multiple Different primers must be used. In this case, certain Sequence sections of the totalsequez as natural PBSs for specific Serve primers. This embodiment is especially for the investigation of already known SNP sites suitable.

In another embodiment is it for reasons favorable to the simplification of the analysis, if a single primer binding site exists in all NSKFs is. According to one preferred embodiment Therefore, according to the invention, the primer binding sites become the NSKFs extra introduced. That way you can Primers with a uniform structure can be used for the reaction.

in the The following will be this embodiment described in detail.

The Composition of the primer binding site is not restricted. Your Length is preferably between 20 and 50 NTs. The primer binding site may be a functional Carrying group for the immobilization of the NSKF. This functional group can e.g. to be a biotin group.

When example for the introduction In the following, a single primer binding agency will be the Ligation and nucleotide tailing to DNA fragments described.

a) Ligation:

there becomes a double-stranded one Oligonucleotide complex used with a Primerbindungsstelle. This comes with commercially available Ligases ligated to the DNA fragments ("Molecular cloning" 1989 J. Sambrook et al., Cold Spring Harbor Laboratory Press). It is important that only one Primer binding site is ligated to the DNA fragment. That achieved one e.g. by a modification of one side of the oligonucleotide complex on both strands. The modifying groups on the oligonucleotide complex can be immobilized serve. The synthesis and modification of such oligonucleotide complex can be carried out according to standardized regulations. For synthesis can e.g. the DNA Synthesizer 380A Applied Biosystems. Oligonucleotides of a particular composition with or without Modifications are also commercially available as a custom synthesis, e.g. from MWG-Biotech GmbH, Germany.

b) nucleotide tailing:

Instead of The ligation with an oligonucleotide can be done with a terminal Deoxynucleotidyltransferase multiple (e.g., between 10 and 20) nucleoside monophosphates to the 3'-end of a attach ss DNA fragments ("Molecular Cloning" 1989 J. Sambrook et al. Cold Spring Harbor Laboratory Press, "Method in Enzymology" 1999 v.303, p.37-38), e.g. several Guanosine monophosphates ((G) n-tailing called). The resulting fragment becomes the binding of the primer, used in this example of a (C) n primer.

Single-stranded preparation

For the sequencing reaction become single-stranded NSKFs needed. If the starting material is in double-stranded form, there there are several ways from double-stranded DNA is a single-stranded one Form (e.g., heat denaturation or alkali denaturation) ("Molecular Cloning" 1989 J. Sambrook et al. Cold Spring Harbor Laboratory Press).

Primer for the sequencing reaction

This has the function to start at a single point of the NSKF enable. He binds to the primer binding agency in the NSKF. The composition and the length of the primer are not restricted. Except the Start function, the primer can also take over other functions, such as. to create a connection to the reaction surface. Primer should so long and composition of the primer binding site, that the primer the start of the sequencing reaction with the respective Polymerase allows.

at the use of different, for example, of course, in the original one Overall sequence of occurring Primerbindungsstellen be those for the respective Primary binding site sequence-specific primer used. In this Case is for the sequencing used a primer mixture.

at a uniform, for example by ligation to the NSKFs coupled primer binding site becomes a uniform primer used.

Preferably is the length of the primer between 6 and 100 NTs, optimally between 15 and 30 NTs. The primer can carry a functional group that is used for immobilization of the NSKF, for example, such a function group is one Biotin group (see section Immobilization). She should do the sequencing do not bother. The synthesis of such a primer can e.g. with the DNA synthesizer 380 A Applied Biosystems or as an order synthesis from a commercial supplier, e.g. MWG-Biotech GmbH, Germany).

Of the Primer is in one embodiment on the surface fixed as described in this application. The primer or the primer mix is mixed with NSKFs under hybridization conditions which selectively delivers it to the primer binding site of the NSKF let bind. This primer annealing can be done before (1) while (2) or after (3) binding of the NSKFs to the surface. The optimization of hybridization conditions depends on the exact structure the primer binding site and the primer and can be according to Rychlik et al. Calculate NAR 1990 v.18 p.6409. Hereinafter These hybridization conditions are considered as standardized hybridization conditions designated.

If one for all NSKFs common primer binding site of known structure For example, by ligation is introduced, primers with uniform Structure are used. The primer binding site can be attached to her 3'-end a functional Wear group, e.g. serves for immobilization. For example this group is a biotin group. The primer has a primer binding site complementary Structure. Fixation of NSKF primer complexes to the surface (binding or immobilization of NSKFs).

aim immobilization is to NSKF-primer complexes a suitable flat surface in a way that fix a cyclic enzymatic Sequencing reaction can proceed. This can be done, for example Binding of the primer (s.o.) or of the NSKF carried to the surface.

• 1) Die NSKF-Primer-Komplexe können zunächst in einer Lösung durch Hybridisierung (Annealing) gebildet und anschließend an die Oberfläche gebunden werden.
• 2) Primer können zunächst auf einer Oberfläche gebunden werden und NSKFs anschließend an die gebundenen Primer hybridisiert werden, wobei NSKF-Primer-Komplexe entstehen (NSKFs indirekt an die Oberfläche gebunden)
• 3) Die NSKFs können zunächst an die Oberfläche gebunden werden (NSKFs direkt an die Oberfläche gebunden) und im anschließenden Schritt die Primer an die gebundenen NSKFs hybridisiert werden, wobei NSKF-Primer-Komplexe enstehen.

The order of steps in fixing NSKF primer complexes may be variable:

• 1) The NSKF primer complexes can first be formed in a solution by hybridization (annealing) and then bound to the surface.
• 2) primers can first be bound to a surface and then NSKFs bound to the which primers are hybridized to form NSKF primer complexes (NSKFs indirectly bound to the surface)
• 3) The NSKFs can first be bound to the surface (NSKFs bound directly to the surface) and in the subsequent step, the primers are hybridized to the bound NSKFs resulting in NSKF primer complexes.

The Immobilization of NSKFs to the surface can therefore be achieved by direct or indirect binding.

If the fixation of the NSKF primer complexes on the surface over the This can be done, for example, by binding the NSKFs take place at one of the two ends of the chain. This can be done through appropriate covalent, affine or other bonds can be achieved. Examples for fixation of nucleic acid chains are listed in this application. Further examples of the immobilization of nucleic acids are (McGall et al U.S. Patent No. 5,41,2087, Nikiforov et al., US Pat Pat. No. 5610287, Barrett et al. US Pat. No. 5482867, Mirzabekov et al. US Patent 5981734, "Microarray biochip technology "2000 M.Schena Eaton Publishing, "DNA Microarrays "1999 M. Schena Oxford University Press, Rasmussen et al. Analytical Biochemistry v.198, P.138, Allemand et al. Biophysical Journal 1997, v.73, p.2064, Trabesinger et al. Analytical Chemistry 1999, v.71, p.279, Osborne et al. Analytical Chemistry 2000, v.72, p.3678, Timofeev et al. Nucleic Acid Research (NAR) 1996, v.24 p.3142, Ghosh et al. NAR 1987 v.15 p.5353, Gingeras et al. NAR 1987 v.15 5.5373, Maskos et al. NAR 1992 v.20 p.1679).

to further reduction of non-specific binding of labeled components to the surface can blocking substances are used. Such substances are known to the skilled person. Often, buffer solutions of proteins (e.g. Serum albumin solution) used as blocking reagents. Also polymer solutions, e.g. Moviol or Polyethylene glycol solution (Molecular weight 10,000) is used.

Choice of polymerase

When In principle, polymerases are suitable for all DNA-dependent DNA polymerases without 3'-5 'exonuclease activity (DNA replication "1992 ed. A.Kornberg, Freeman and company NY), e.g. modified T7 polymerase type "Sequenase Version 2 "(Amersham Pharmacia Biotech), 3'-5 'exonuclease free Klenow fragment of DNA polymerase I (Amersham Pharmacia Biotech), Polymerase Beta of various origins ("Animal Cell DNA Polymerases" 1983, Fry M., CRC Press Inc., commercially available in Chimerx) thermostable polymerases such as Taq polymerase (GibcoBRL), proHATM polymerase (Eurogentech).

polymerases with 3'-5 'exonuclease activity can be used (e.g., Klenow fragment the E. coli polymerase I), if reaction conditions are chosen, suppress the existing 3'-5 'exonuclease activity, such as e.g. a low pH (pH 6.5) in the Klenow fragment (Lehman and Richardson, J. Biol. Chem. 1964 v. 239 p.233) or addition of NaF for the installation reaction. Another possibility is the use of NTs * with a phosphorothioate compound (Kunkel et al., PNAS 1981, v. 78 p. 6734). Incorporated NTs * of the 3'-5 'exonuclease activity of the polymerase not attacked. In the following, all these types of polymers will be mentioned referred to as "polymerase".

Chemistry, general principle

For the sequencing reaction in highly parallel sequence analysis on individual nucleic acid molecules (parallel Analysis of up to 10,000,000 NSKF sequences) is important that every built-in NT * is identified.

• 1) Die Reaktion muss so gesteuert werden, dass die Polymerase NT*s einzeln einbaut (Stop des weiteren Einbaus).
• 2) NT*s müssen einen Farbstoff tragen, der den Anforderungen der Detektion genügt.
• 3) Der Farbstoff muß unter milden Bedingungen abspaltbar sein, so dass weder die NSKF-Primer-Komplexe, noch einzelne Komponenten des Systems beschädigt werden.
• 4) Die Abspaltung muss möglichst schnell und quantitativ erfolgen.
• 5) Der Stop des Einbaus muss reversibel sein und unter milden Bedingungen aufgehoben werden können.

The difficulties in developing suitable NT analogs for the process are based on the following framework conditions:

• 1) The reaction must be controlled in such a way that the polymerase incorporates NT * s individually (stop the further installation).
• 2) NT * s must carry a dye that meets the requirements of detection.
• 3) The dye must be cleavable under mild conditions, so that neither the NSKF primer complexes nor individual components of the system are damaged.
• 4) The splitting off must be as fast as possible and quantitative.
• 5) The stop of installation must be reversible and can be reversed under mild conditions.

A coupled to the base sterically demanding group can be a hindrance lead the further synthesis. Biotin, digoxigenin and fluorescent dyes such as fluorescein, tetramethylrhodamine, Cy3 dye represent examples of such a sterically demanding group (Zhu et al., Cytometry 1997, v.28, p.206, Zhu et al., NAR 1994, v.22, p.3418, Gebeyehu et al., NAR 1987, v.15, 5.4513, Wiemann et al. Analytical Biochemistry 1996, v.234, p.166, Heer et al. BioTechniques 1994 v.16 p.54).

at the sequencing reaction in the method according to the invention are labeled NTs * incubated with a polymerase and nucleic acid chains. The NTs * carry a reversibly coupled to the base sterisch demanding Group. When a reaction mixture containing only modified NTs * in the Reaction is used, then the polymerase can only a single Install NT *. The installation of a next NT * is sterically inhibited. These NTs * thus act as terminators the synthesis. After the removal of the sterically demanding Group can be the next complementary NT * be installed. Because these NTs are not an absolute obstacle to represent another synthesis, but only for the incorporation of another marked NT *, they are called Semiterminatoren.

General structure of the NT *

These Structure is characterized by the fact that at the base over a cleavable linkers are bound to a fluorescent label.

When basis for the NTs * serve deoxynucleoside triphosphates with adenosine (A), guanosine (G), Cytidine (C) and thymidine (T) as nucleoside residue. Instead of thymidine uridine is preferably used as the nucleoside residue. Instead of Guanosine can be used in inosine.

Markers, fluorophores

• a) Die verwendete Detektionsapparatur muß diesen Marker als einziges Molekül gebunden an DNA unter milden Bedingungen (vorzugsweise Reaktionsbedingungen) identifizieren können. Die Farbstoffe haben vorzugsweise große Photostabilität. Ihre Fluoreszenz wird vorzugsweise von der DNA nicht oder nur unwesentlich gequencht.
• b) Der an das NT gebundene Farbstoff darf keine irreversible Störung der enzymatischen Reaktion verursachen.
• c) mit dem Farbstoff markierte NTs* müssen von der Polymerase in die Nukleinsäurekette eingebaut werden.
• d) Bei einer Markierung mit verschiedenen Farbstoffen sollen diese Farbstoffe keine beträchtlichen Überlappungen in ihren Emissionsspektren aufweisen.

Each base is marked with a marker (F) that is characteristic of it. The marker is a fluorescent dye. Several factors influence the choice of fluorescent dye. The choice is not limited if the dye meets the following requirements:

• a) The detection apparatus used must be able to identify this marker as a single molecule bound to DNA under mild conditions (preferably reaction conditions). The dyes preferably have high photostability. Their fluorescence is preferably not or only insignificantly quenched by the DNA.
• b) The dye bound to the NT must not cause irreversible disruption of the enzymatic reaction.
• c) NTs * labeled with the dye must be incorporated by the polymerase into the nucleic acid chain.
• d) When labeled with different dyes, these dyes should not have significant overlaps in their emission spectra.

in the Fluorescent dyes which can be used according to the present invention are in "Handbook of Fluorescent Probes and Research Chemicals "6th ed. 1996, R. Haugland, Molecular Probes assembled with structural formulas. According to the invention preferably the following classes of dyes used as markers: cyanine dyes and their descendants (e.g., Cy2, Cy3, Cy5, Cy7 Amersham Pharmacia Biotech, Wagoner US Pat. No. 5,268,486), rhodamines and their derivatives (e.g., TAMRA, TRITC, RG6, R110, ROX, Molecular Probes, s. Manual), xanthene derivatives (e.g., Alexa 568, Alexa 594, Molecular Probes, Mao et al U.S. Pat. No. 6.130.101). Atto dyes (Attotec GmbH, Siegen, Germany) These dyes are commercially available.

there you can depending on the spectral properties and existing equipment select appropriate dyes. The dyes are added to the linker e.g. via thiocyanate or ester linkage coupled ("Handbook of Fluorescent Probes and Research Chemicals "6th ed. 1996, R. Haugland, Molecular Probes, Jameson et al. Methods in Enzymology 1997 v.278 p. 363, Wagoner Methods in Enzymology 1995 v.246 p.362).

Nature of sterically demanding group

Fluorescent dyes are examples of a bulky group (Zhu et al., Cytometry 1997, v.28, p.206, Zhu et al., NAR 1994, v.22, p.3418, Gebeyehu et al., NAR 1987, v.15 , P.4513, Wiemann et al., Analytical Biochemistry 1996, v.234, p.166, Heer et al., BioTechniques 1994 v.16 p.54). The chemical Structure of this group is not limited provided it does not significantly interfere with the incorporation of the labeled NT * to which it is attached and does not cause irreversible disruption of the enzymatic reaction.

By the cleavage of the linker becomes this sterically demanding group (D) removed after detection of the signal, leaving the polymerase can install another marked NT *.

left

Of the Marker (fluorescent dye) is preferably attached to the base via a Spacers of different lengths, a so-called linker, bound. Preferably, this linker is at one of the sites on the base bound, which does not participate in the base pairing. In the preferred Case are the sites to which the linker is bound: the 4 or 5-position in the pyrimidine ring and the 7-position or 6-position in the Purine ring. Examples of the coupling of a linker to the base can be made following sources (Hobbs et al., US Pat. No. 5,047,519, Khan et al. U.S. Pat. No. 5,821,356, Hanna M. Method in Enzymology 1996, v.274, p.403, Zhu et al. NAR 1994 v.22 p.3418, Herman et al. Methods in Enzymology 1990 v.184 p.584, J.L. Ruth et al. Molecular Pharmacology 1981 v.20 p.415, L. Ötvös et al. NAR 1987 v.15 p.1763, G.E. Wright et al. Pharmac Ther. 1990 v.47, p. 447, "Nucleotides Analogs; Synthesis and Biological Function "K.H. Scheit 1980, Wiley-Interscience Publication, "Nucleic acid chemistry "Ed. L.B. Townsend, v.1-4, Wiley-Interscience Publication, "Chemistry of Nucleosides and Nucleotides "Ed. L.B. Townsend, v.1-3, Plenary Press).

The whole length of the linker may vary. It corresponds to the number of chain atoms and is preferably between 3 and 50. Optimally, it is between 4 and 10 atoms. The chemical composition of the linker is not limited, provided that it remains stable under reaction conditions and does not interfere with caused enzymatic reaction.

Splittable compound, cleavage

Of the Left carries a cleavable or cleavable group. This fissile Connection allows the removal of the marker and the steric obstruction at the end every cycle. Your choice is not limited, unless under the conditions the enzymatic sequencing reaction remains stable, not irreversible disorder caused the polymerase and cleaved under mild conditions can be. Under "mild Conditions "are to understand such conditions that do not destroy the NSKF-primer complex, wherein e.g. the pH is preferably between 3 and 11, the temperature between 0 ° C and a temperature value (x). This temperature value (x) depends on the Tm of the NSKF primer complex (Tm is "melting point") and is used, for example, as Tm (NSKF primer complex) minus 5 ° C calculated (e.g., Tm is 47 ° C, then the maximum temperature is 42 ° C; under these conditions Particularly suitable ester, thioester, disulfide compounds and photolabile compounds as cleavable compounds).

Preferably belongs said compound to be chemically or enzymatically cleavable or photolabile compounds. As examples of chemically cleavable Groups are preferred ester, thioester and disulfide compounds ( "Chemistry of protein conjugation and crosslinking "Shan S. Wong 1993 CRC Press Inc., Herman et al. Method in Enzymology 1990 v.184 p.584, Lomant et al. J. Mol. 1976 v.104 243, "Chemistry of carboxylic acid and esters "S.Patai 1969 Interscience Publ.). Examples of photolabile compounds can in following references: "Protective groups in organic synthesis" 1991 John Wiley & Sons, Inc., V. Pillai Synthesis 1980 p.1, V. Pillai Org. Photochem. 1987 v.9 p.225, Dissertation "New photolabile protecting groups for light-directed oligonucleotide synthesis "H.Giegrich, 1996, Konstanz, Dissertation" Neue photolabile Protecting groups for light-directed oligonucleotide synthesis "S.M. Buehler, 1999, Konstanz).

The Position of the cleavable compound / group in the linker is preferred not more than 10 atoms away from the base, more preferably not more than 3 atoms. Particularly preferred is the cleavable Compound or group directly at the base.

Of the Cleavage and removal step is present in each cycle and must under mild conditions (see above) so that the nucleic acids are not damaged or modified.

The cleavage proceeds preferably chemically (eg in mild acidic or basic environment for an ester compound or by addition of a reducing agent, eg dithiothreitol or mercaptoethanol (Sigma) in the cleavage of a disulfide compound), or physically (eg by illuminating the surface with light a particular wavelength for the cleavage of a photolabile group, thesis "New photolabile Protecting groups for light-directed oligonucleotide synthesis "H. Giegrich, 1996, Konstanz).

To the cleavage remains at the base a linker residue. By the split can arise on the linker radical reactive group, such as a Mercapto. Such groups can be chemically modified as needed. Many examples of modification of mercapto groups are known ("Chemistry of protein conjugation and crosslinking "Shan S. Wong 1993 CRC Press Inc.) e.g. Disulfide, such as dithiodinitro-phenol, dithiomercaptoethanol, Cystamines, e.g. or iodoacetate compounds, e.g. Iodoacetamide, iodoacetate or maleimides).

Some examples for Syntheses of nucleotide analogues are shown in Example 7.

Colored coding scheme, Number of dyes

• a) vier verschieden markierten NT*s
• b) zwei verschieden markierten NT*s
• c) einem markierten NT*
• d) zwei verschieden markierten NT*s und zwei unmarkierten NTs,

d.h.

• a) Man kann alle 4 NTs mit verschiedenen Farbstoffen markieren und alle 4 gleichzeitig in die Reaktion einsetzten. Dabei erreicht man die Sequenzierung einer Nukleinsäurekette mit einer minimalen Anzahl von Zyklen. Diese Variante der Erfindung stellt allerdings hohe Anforderungen an das Detektionssystem: 4 verschiedene Farbstoffe müssen in jedem Zyklus identifiziert werden.
• b) Zur Vereinfachung der Detektion kann eine Markierung mit zwei Farbstoffen gewählt werden. Dabei werden 2 Paare von NTs gebildet, die jeweils verschieden markiert sind, z.B. A und G tragen die Markierung "X", C und U tragen die Markierung "Y". In die Reaktion in einem Zyklus (n) werden 2 unterschiedlich markierte NTs gleichzeitig eingesetzt, z.B. C* in Kombination mit A*, und im darauffolgenden Zyklus (n+1) werden dann U* und G* zugegeben.
• c) Man kann auch nur einen einzigen Farbstoff zur Markierung aller 4 NTs* verwenden und pro Zyklus nur ein NT* einsetzen.
• d) In einer technisch vereinfachten Ausführungsform werden pro Zyklus zwei unterschiedlich markierte NT*s eingesetzt und zwei unmarkierte NTs (sogen. 2NT*s/2NTs-Methode). Diese Ausführungsform kann verwendet werden, um Varianten (z.B. Mutationen, oder alternativ gespleißte Gene) einer bereits bekannten Sequenz zu ermitteln.

One cycle can be done with:

• a) four differently labeled NT * s
• b) two different marked NT * s
• c) a marked NT *
• d) two differently labeled NT * s and two unlabelled NTs,

ie

• a) One can mark all 4 NTs with different coloring materials and insert all 4 at the same time in the reaction. It achieves the sequencing of a nucleic acid chain with a minimum number of cycles. However, this variant of the invention makes high demands on the detection system: 4 different dyes must be identified in each cycle.
• b) To simplify the detection, a label with two dyes can be selected. In this case, two pairs of NTs are formed, which are each marked differently, eg A and G carry the mark "X", C and U carry the mark "Y". In the reaction in one cycle (s) 2 differently labeled NTs are used simultaneously, eg C * in combination with A *, and in the next cycle (n + 1) then U * and G * are added.
• c) It is also possible to use only one dye to mark all 4 NTs * and use only one NT * per cycle.
• d) In a technically simplified embodiment, two differently marked NT * s are used per cycle and two unmarked NTs (so-called 2NT * s / 2NTs method). This embodiment can be used to detect variants (eg, mutations, or alternatively spliced genes) of an already known sequence.

In an embodiment of the method will be several modifications of a nucleotide type used.

For example Several modifications of dCTP nucleotides are used. These Modifications can in one embodiment be present in the same reaction mixture. In another embodiment become single variants of the modified nucleotides with the progress the sequencing changed. For example, at the beginning of a Sequencing reaction nucleotide modifications used with a short linker, for example Example 7C or 7D. In the course of sequencing then nucleotide analogs with longer Used linkers, for example Example 7A. At the end of the sequencing can then nucleotides are used with an even longer linker, For example, Example 7B. In one embodiment of the method it is advantageous for the first 25% of the corresponding cycles with a nucleotide analog perform, the next 25% of the corresponding cycles with another, etc. The replacement nucleotides can also be performed in a different order, for example, after 10% of all cycles, after 20%, etc., nucleotides may be present with, for example, ever longer Linker between a base and a dye can be used. The exchange of nucleotide analogues can also be performed after a fixed Cycle number. For example, the first 10 cycles with a nucleotide analogue performed then, for example, a different nuclide analogue is used. After 30 cycles, another nucleotide analogue is used and after 60 cycles, a still further nucleotide analog is used.

Of the Advantage of such exchange of nucleotides used in that with increasing number of already incorporated nucleotides grows the degree of modification of the nucleic acids so that nuclides with a shorter one Left are installed worse than those with a longer one. By replacing the nucleotides used thus longer sequence stretches can be analyzed become. Such an exchange can be modified for all used Nucleotides take place or only for a part thereof.

All in all play the size that Charge and the chemical structure of the marker, the length of the fissile Linker and the linker residue as well as the choice of polymerase an important role. You decide together if the marked NT * is incorporated by the polymerase in the growing nucleic acid chain, and whether by the installation of the next marked NT * is prevented.

Similar to when replacing used nucleotide modifications can also exchanged polymerases with the progress of sequencing become.

So for example the first 10% of cycles are performed with a Klenow polymerase, then, for example, Sequenase 2 is used. After 50% of all Cycles are used for example Vent Exo minus and for the last 20% of all cycles use Pwo polymerase. The exchange Polymerases can also be carried out after a fixed cycle number. For example, you can the first 10 cycles are carried out with a Klenow polymerase, then, for example, Sequenase 2 can be used. After another For example, Vent Exo minus is used for 10 cycles and after another 20 cycles Pwo polymerase is used. Generally Let's start with polymerases that are less tolerant Modifications are and with the increasing number of cycles, and thus with the increasing degree of modification of the DNA, changing into polymerases, which are more tolerant to modifications. Differences in polymerases with respect to nucleotide modifications are in Augustin et al. "Progress towards singlemolecule sequencing: enzymatic synthesis of nucleotide-specifically labeled DNA " Biotechnol 2001, v. 86, 289-301.

There with the progress of the sequencing also the length of the newly formed double strand rises, can also the reaction conditions with the progress of the sequencing changed For example, the temperature can be raised.

Now follow some examples of cyclic reactions with those on the Fixed surface of the invention Nucleic acids.

6.1 Installation reaction of labeled nucleotides on the nucleic acid primer complexes bound on the surface are.

• a) Inkubation einer Lösung mit einer oder mehreren Polymerase und einem oder mehreren markierten Nukleotiden (Polymerase-Nukleotid-Gemisch) mit der festen Phase unter Bedingungen, die eine Einbaureaktion an den gebildeten Primer-Matrize-Komplexen zulassen,
• b) Waschen der festen Phase
• c) Detektion der Signale von eingebauten markierten Nukleotiden, wobei relative Koordinaten einzelne Signale identifiziert werden
• d) Entfernung der Signale von eingebauten Nukleotiden,
• e) Waschen der festen Phase
• f) gegebenenfalls Wiederholung der Schritte (a) bis (e)

As indicated above, nucleic acids can be bound to the regenerated surfaces of the solid phase, specifically participating in hybridization reactions with complementary oligonucleotides, and the unspecific binding of labeled components such as nucleic acids or nucleotides remains very low. In the following examples it is shown that the solid phase prepared according to the invention is suitable for the analysis of individual nucleic acid molecules, wherein it has a very low unspecific binding of labeled components. Initially, extensible nucleic acid chain primer complexes are formed on the surface. Subsequently, a cyclic reaction is carried out, which essentially concludes the following steps:

• a) incubation of a solution with one or more polymerase and one or more labeled nucleotides (polymerase-nucleotide mixture) with the solid phase under conditions which allow a incorporation reaction on the formed primer-template complexes,
• b) washing the solid phase
• c) detection of signals from incorporated labeled nucleotides, relative coordinates of which are identified as individual signals
• d) removal of signals from incorporated nucleotides,
• e) washing the solid phase
• f) optionally repeating steps (a) to (e)

Out the order of the detected signals with identical x, y coordinates will connect the sequence is reconstructed (WO 02088382).

The Number of steps may vary. In some applications, such as in "minisequencing" or primer extension, see above, only one nucleotide is incorporated. In a sequencing several cycles are carried out their number is between 5 and 100, for example.

6.1 Sequencing reaction on an oligonucleotide

In This example is a highly parallel sequencing reaction an oligonucleotide performed. The following components were used:

• dUTP-SS-Cy3, dCTP-SS-Cy3 (beide synthetisiert nach Vorschrift in WO 02088382, sieh 7f-1 und 7f-4, wobei die Aminogruppe am Linker mit einem Cy3-Farbstoff modifiziert ist, s. Beispiel 2 derselben Anmeldung oder auch nach Prozedur im Beispiel 7A).
• dATP, dGTP (Sigma-Aldrich)
• Polymerase: Klenow Exo minus (Amersham Bioscience)
• Matrize: Oligo-31 (Sequenz s. Tabelle 18)
• Primer: T7-19 (Sequenz s. Tabelle 18)
• Puffer und Arbeitslösungen: Einbaupuffer: 50 mmol/l Tris-HCl, pH 8.7, 50 mmol/l NaCl, 1 mmol/l MgCl₂ Spaltungspuffer: TCEP 100 mmol/l, pH 8.0 Alkylierungspuffer: Iodacetamid 100 mmol/l in 50 mmol/l Borat-Puffer pH 9.0 Waschpuffer entspricht dem Einbaupuffer
• Lösung "U" 1 μmol/l dUTP-SS-Cy3 in Einbaupuffer + Polymerase 1 Unit in 50 μl
• Lösung "C" 1 μmol/l dCTP-SS-Cy3 in Einbaupuffer + Polymerase 1 Unit in 50 μl
• Lösung "A,G" 0,1 μmol/l dATP und dGTP im Einbaupuffer + Polymerase 1 unit in 50 μl

The surface of the solid phase carries anchor oligonucleotides. It was produced as described in Example 5.1.2.

• dUTP-SS-Cy3, dCTP-SS-Cy3 (both synthesized according to protocol in WO 02088382, see 7f-1 and 7f-4 wherein the amino group on the linker is modified with a Cy3 dye, s. Example 2 of the same application or also according to the procedure in Example 7A).
• dATP, dGTP (Sigma-Aldrich)
• Polymerase: Klenow Exo minus (Amersham Bioscience)
• Template: oligo-31 (sequence see Table 18)
• Primer: T7-19 (sequence see Table 18)
• Buffer and working solutions: Buffer: 50 mmol / l Tris-HCl, pH 8.7, 50 mmol / l NaCl, 1 mmol / l MgCl ₂ Cleavage buffer: TCEP 100 mmol / l, pH 8.0 Alkylation buffer: iodoacetamide 100 mmol / l in 50 mmol / l Borate buffer pH 9.0 Wash buffer corresponds to the built-in buffer
• Solution "U" 1 μmol / l dUTP-SS-Cy3 in incorporation buffer + polymerase 1 unit in 50 μl
• Solution "C" 1 μmol / l dCTP-SS-Cy3 in incorporation buffer + polymerase 1 unit in 50 μl
• Solution "A, G" 0.1 μmol / l dATP and dGTP in the incorporation buffer + polymerase 1 unit in 50 μl

polymerase was modified in this example with iodoacetamide as follows: to 75 μl the aliquot of Klenow Exo minus polymerase (Amersham-Bioscience, 750 unit Klenow exo minus) was iodoacetamide (Sigma-Aldrich) to added to concentration of 20 mmol / l and at RT for 30 min touched. Further notes for the modification is given in Annex 1:

To the anchor oligonucleotides an oligo-31 was hybridized:

A 100 nmol / l solution of oligo 31 in the built-in buffer (20 μl) was for Brought into contact with the surface of the solid phase at RT for 5 min, subsequently became the surface with the built-in buffer (100 μl) washed. Subsequently became the

Primer T7-19 to the primer binding site hybridized in Oligo 31:

A 10 nmol / l solution of T7-19 in the built-in buffer (20 μl) was for Brought into contact with the surface of the solid phase at RT for 5 min, subsequently became the surface with the built-in buffer (100 μl) washed.

For detection, the optical system shown above was used, with several positions on the surface being analyzed. One position has an area of 4800 μm ² . The signal was counted with software that can identify fluorescence signals from single molecules. The sequencing method is described in detail in the applications WO 02088382, WO 03031947, which describes the use of the novel solid phase for such methods. For reproducible finding of the same positions on the solid phase over several cycles, an alignment pattern was applied. In this case, the pattern consisted of ink particles that had absorbed from an aqueous solution onto the surface of the solid phase. These particles allow optical readjustment of the positions of the surface and the recovery of individual sequencing sites (primer-plasmid complexes).

The Number of signals from individual labeled nucleic acids incorporated into the nucleic acid Nucleotide molecules on individual positions is presented in Table 15.

It a total of 20 cycles were performed.

cycle (A) closes following steps:

• 1) the surface of the solid phase with the fixed primer-template complexes were incubated with cleavage buffer (10 μl) for 5 min incubated at RT, then washed with the wash buffer, then for 5 min at RT with alkylation buffer (20 μl) washed (to block free SH groups) and then with wash buffer washed.
• 2) it was the solution "A, G" (10 ul) on the surface and incubated at RT for 5 min, so that optionally one Assembly reaction of dATP and or dGTP can take place. Subsequently was the surface washed with the washing buffer.
• 3) detection

cycle (U) closes following steps:

• 1) the surface of the solid phase with the fixed primer-template complexes was treated with solution "U" 5 incubated at RT and then washed with the washing buffer.
• 2) detection

cycle (C) closes following steps:

• 1) the surface of the solid phase with the fixed primer-template complexes was treated with solution "C" 5 incubated at RT and then washed with the washing buffer.
• 2) detection

Table 15 Summation Signal, 01330 MFC: Channel B,

The number of detected signals changes in individual cycles. When signals from single nucleotide molecules having the same coordinates are ordered in the order of their appearance from cycle 1 to cycle 20, a distribution of orders of the signals results. These sequences (sequence raw data) were reconstructed for position 2 and 3 and are in the 9a and 9b presents. Since over 3500 oligonucleotides were sequenced in parallel at one position, sequences having identical sequences were pooled. The number of identical sequence orders can be seen to the right of each sequence. Each letter represents the detected signal: "U" stands for incorporated labeled dUMP-SS-Cy3, "C" stands for incorporated labeled dCMP-SS-Cy3, "A" stands for the surface remaining signals after the cleavage step, hyphens between individual letters mean that no signal has been assigned to corresponding coordinates in the respective cycle.

In In this example, only two labeled and two unlabeled nucleotides were used used. The sequencing reaction proceeds differently on individual oligonucleotide molecules fast, making it a distribution of actually measured Sequences is coming. The sequences are shown in Table 20 (see Appendix 3) in different lengths summarized, corresponding to the number of measured signals with the same coordinates. You can see columns with the name length 1, length 2 ... length 5.

The to be determined sequence "-U-U-U-U-C-" was 215 times detected in both positions. Other sequences are only partial extended or erroneous sequences.

• 1) an der erfindungsgemäßen Oberfläche kann eine hochparallele Sequenzierungsreaktion durchgeführt werden, wobei Signale von einzelnen Molekülen detektiert werden.
• 2) Die unspezifische Bindung von markierten Komponenten, wie beispielsweise dCTP-SS-Cy3 ist sehr gering: bei Inkubationsschritten, die keinen Einbau von dCTP-SS-Cy3 zulassen (die Sequenz hat die potenzielle Stelle noch nicht erreicht), beträgt die unspezifische Bindung weniger als 10 Ereignisse pro 100 μm² (s. z.B. Zyklen 1, 5, 8, 11).

In connection with this application, the following facts are of great importance:

• 1) a highly parallel sequencing reaction can be carried out on the surface according to the invention, whereby signals of individual molecules are detected.
• 2) The non-specific binding of labeled components, such as dCTP-SS-Cy3, is very low: in incubation steps that do not permit the incorporation of dCTP-SS-Cy3 (the sequence has not yet reached the potential site), the non-specific binding is less as 10 events per 100 μm ² (eg cycles 1, 5, 8, 11).

In another example of sequencing the oligonucleotides having the surface of the invention, the concentration of oligo-31 bound to the solid phase via the linker was reduced by half. All other steps were carried out identically as in the example described above. It can be seen in Table 16 that the number of signals resulting from incorporation of labeled nucleotides is only half of the signals in the corresponding step in the other example (Table 15). However, the non-specific binding of labeled nucleotides is just as low (below 10 events per 100 μm ² ).

Table 16 Summation Signal, 01330 MFC: Channel A,

In similar wise also other surfaces of the invention solid phase can be used.

6.2 Reaction at the M-13 plasmid

In This example shows that with the solid according to the invention Phase a sequence-dependent Incorporation reaction on long nucleic acid chain fragments (NSKFs) carried out can be. The nonspecific binding of NSKFs to the surface is low.

The solid phase was prepared as in Example 5.1.3 so that oligonucleotides having a free 3 'group are fixed to the surface of the solid phase and can serve as primers for the extension reaction NEN. To these oligonucleotides were hybridized fragments of the M-13 plasmid which were provided with a poly-dT stretch for hybridization.

The reaction proceeded in several steps, individual steps shown in Table 17. For detection, the optical system shown above was used, with several positions on the surface being analyzed. One position has an area of 4800 μm ² . The signal analysis was performed using software that can identify fluorescence signals from single molecules. The process is described in detail in applications (WO 02088382, WO 03031947), which describes the use of the novel solid phase for such processes.

In detail:

Material Preparation

One single-stranded M-13 plasmid, approximately 7.4 kb in length, (Amersham Bioscience) in concentration 100 ng / μl, 100μl, in 20mM Tris-HCl buffer, pH 8.5, was sonicated (400 watts) 10 sec fragmented. Subsequently was a "tailing" reaction with a Mixture of dTTP (100 μmol / l) and dUTP-Cy3 (Amersham-Bioscience), 1 μmol / L, with the terminal deoxynucleotidyltransferase (Amersham Bioscience), as in WO 02088382 or in "Molecular cloning" 1989, Ed. Maniatis, Cold Spring Harbor Laboratory is shown. The reaction product was cleaned with Qiaquick kit (Qiagen) for PCR products. The purified plasmid DNA fragments (dissolved in water) were heated to 95 ° C and cooled to RT.

Fixation to the surface

Fragments of M-13 plasmid were diluted to concentration of 5 ng / μl diluted in 50 mmol / l Tris-HCl buffer, 50 mmol / l NaCl, 1 mmol / l MgCl ₂ and in contact with the solid phase for 30 min brought. Hybridization of M-13 fragments with poly-dT tracts to the surface-fixed oligo-dA50 was performed. The bond to the surface was between 100 and 400 strands per 100 μm ² . In control experiments, M-13 fragments were brought into contact with the surfaces of the solid phases which did not contain oligo-dA50 and were prepared according to the procedure in Example 1.1, 4.1 or 5.1.2. In these control experiments only a small non-specific binding of M-13 fragments to the solid phase was detected (below 30 signals per 100 μm ² ).

The Signals at the M-13 fragments are from the tailing-built dUTP-Cy3 molecules. To the detection of the density of the M-13 fragments became these dyes by irradiation of the surface faded, so they later Steps do not emit fluorescence signals.

to reproducible finding of the same positions on the solid Phase over several cycles an adjustment pattern was applied. In this case The pattern consisted of ink particles, which consisted of an aqueous solution on the surface have absorbed the solid phase. These particles allow an optical readjustment of the positions of the surface and the recovery of individual sequencing sites (primer-plasmid complexes).

The Sequencing reaction was performed with dUTP and dCTP analogs which Example 7B (dUTP-SOS-Cy3 and dCTP-SOS-Cy3). A similar Sequencing reaction can also be performed with other nucleotides which are synthesized as shown in Example 7 or WO 02088382.

When Polymerase was Klenow exo - (Amersham Bioscience) in concentration 1 unit / 50 μl.

Buffers and working solutions:

• Installation buffer: 50 mmol / l Tris-HCl, pH 8.7, 50 mmol / l NaCl, 1 mmol / l MgCl ₂
• Cleavage buffer: mercaptoethanol 100 mmol / l, in the built-in buffer
• Block buffer: iodoacetamide 100 mmol / l in the incorporation buffer
• Wash buffer corresponds to the built-in buffer.
• Solution "U" 1 μmol / l dUTP-SOS-Cy3 in built-in buffer + polymerase 1 unit in 50 μl
• Solution "C" 1 μmol / l dCTP-SOS-Cy3 in built-in buffer + polymerase 1 unit in 50 μl
• Solution "A, G" 0.1 μmol / l dATP and dGTP in the built-in buffer + polymerase 1 unit in 50 μl

All Reaction steps (incorporation reaction, cleavage) were carried out at room temperature (RT) for 5 min.

It a total of 104 cycles were performed.

cycle (T) Close following steps:

• 1) Hybridization of fragments of M-13 plasmid
• 2) detection
• 3) bleaching the signals from the incorporated labeled nucleotides.

cycle (A) closes following steps:

• 1) the surface of the solid phase with the fixed primer-template complexes were incubated with cleavage buffer (10 μl) for 5 min incubated at RT and then washed with the washing buffer, then block buffer for 5 min treated at RT.
• 2) the solution "A, G" (10 μl) was applied to the surface and incubated at RT for 5 min, so that optionally one Assembly reaction of dATP and or dGTP can take place. Subsequently was the surface washed with the washing buffer.
• 3) detection

cycle (a) closes following steps:

• 1) the surface of the solid phase with the fixed primer-template complexes were incubated with cleavage buffer (10 μl) for 5 min incubated at RT and then washed with the washing buffer, then block buffer for 5 min treated at RT.
• 2) detection

cycle (U) closes following steps:

• 1) the surface of the solid phase with the fixed primer-template complexes was incubated with the solution "U" for 5 min at RT and then with Washed the washing buffer.
• 2) detection

cycle (C) closes following steps:

• 1) the surface of the solid phase with the fixed primer-template complexes was incubated with the solution "C" for 5 min at RT and then with Washed the washing buffer.
• 2) detection

(deviations of these steps are indicated by italics)

Result:

The changes the number of signals from individual incorporated labeled nucleotides can be seen in Table 17.

Table 17 Summation Signal, 00979 MFC: Channel A,

In The table contains lines for cyclic steps, the columns for positions. The numbers give each measured number of fluorescence signals from individual molecules per one position in the respective cycle. The mean means the average number of signals from 4 positions.

In This example should evaluate the data based on the fact the sequence dependence of Incorporation reaction of labeled nucleotides and the small nonspecific Limit binding of labeled nucleotides to the surface during analysis. The Sequences are not evaluated in this example.

Evaluation:

It became a sequence dependent Reaction observed. By variations in the order of addition labeled nucleotides are shown to be sequence specific is: by repeated successive addition of nucleotides the same way, the incorporation of these nucleotides decreases since to disposal saturated passages (see cycles 16 to 28 and 30 to 35). In the following The installation was resumed as all sequences potentially waiting for supply of labeled nucleotides.

When all potential sites are saturated for incorporation, only nonspecifically surface-bound labeled nucleotides are observed. In the calculation of the number of nonspecifically bound labeled nucleotides, the average number of signals remaining after the cleavage is subtracted. In cycles 28, 34 and 68 it can be seen that the number of signals from labeled nucleotides is below 40 per 100 μm ² . In the cycles 28 and 34 there is the possibility of an incorporation reaction (the polymerase is in the solution). In cycle 68, only dUTP-SOS-Cy3 was added without polymerase, correspondingly fewer signals are seen.

D

= Dichte der unspezifisch gebundenen markierten Komponenten

N

= Zahl der Signale im Zyklus N mit markierten Nukleotiden

A

= Zahl der Signale im vorangegangenen Abspaltungszyklus

4800

= Fläche einer Position, μm

100

= Bezugsfläche für die Dichteberechung, μm

The formula for calculating the density of unspecifically bound labeled components: D = ((NA) × 100) / 4800 In which:

D

Density of unspecifically bound labeled components

N

= Number of signals in cycle N with labeled nucleotides

A

= Number of signals in the previous cleavage cycle

4800

= Area of a position, μm

100

= Reference surface for the density calculation, μm

Example 7

Synthesis of mofected nucleotides

Examples for syntheses of nucleotide analogs and their cleavage conditions, which in one Sequencing can be used, s. Example 6.

Analogous to these syntheses can also other nucleotide analogues are synthesized. As starting substances can for example, for Syntheses 7A-7C: 7-deaza-ATP-7-propargylamine, 7-deaza-dATP-7-propargylamine, 7-deaza-ddATP-7-propargylamine, 7-deaza-GTP-7-propargylamine, 7-deaza-dGTP-7-propargylamine, 7-deaza-ddGTP-7-propargylamine, UTP-5-propargylamine, dUTP-5-propargylamine, ddUTP-5-propargylamine, CTP-5-propargylamine, dCTP-5-propargylamine, ddCTP-5-propargylamine, etc. used will be available (available from Chembiotech, Münster, Deuschland, Jena Bioscience, Jena, Germany). In the further example, for example following precursors of other nucleotide analogs similar to Synthesis 7D on 5-iodo-2'-deoxyuridine 7-iodo-7-deaza-2'-deoxyadenosine, 7-iodo-7-deaza-2'-deoxy-guanosine, 5-iodo-2'-deoxycytidine (available from Chembiotech, Münster, Deuschland, Jena Bioscience, Jena, Germany).,

Separation Methods:

Thin Layer Chromatography, DC:

Analytical: "DC aluminum foils 20 × 20 cm silica gel 60 F 254 "(VWR), coated with fluorescent indicator. Visualization is done by means of UV light. Eluent ethanol-water mixture (70:30) (eluent, LM 1) or ethanol-water mixture (90:10) (LM 2).

synthetically: Silica gel glass plates with collecting layer (VWR). LM 1 or LM 2.

Reverse phase chromatography (RP-chromatography), RP-18:

C-18 Material (Fluka), column volume 10ml, water-methanol gradient. Fractions per 1ml were collected and analyzed with UV-Vis spectrometer. Fractions with similar Spectra were pooled and lyophilized.

HPLC columns with similar Material can used (Sigma), gradient: aqueous buffer: acetonitrile (Acetate buffer: acetonitrile).

Ion exchange chromatography

DEAE-cellulose (VWR), gradient NH ₄ HCO ₃ 20mmol / l - 1mol / l, fractions were collected under UV / Vis control and pooled on the same spectra.

Material:

dUTP-AA (dUTP-5-Allylamine, Jena-Bioscience,) dCTP-PA (dCTP-5-propargylamine, Jena-Bioscience), PDTP-NHS (3- (2-pyridinyl-dithio) -propionic acid-N-hydroxysuccinimidyl-ester, Sigma-Aldrich-Fluka, Taufkirchen, Germany, (hereinafter referred to as Sigma or Aldrich or Fluka)), CDI (1,1'-carbonyldiimidazole, Sigma), Cy3 (dye, Amerscham Bioscience, Freiburg, Germany, further as Amersham Bioscience ), Cy3-NHS (Cy3-N-) hydroxysuccinimidyl ester, Amerscham Bioscience), MEA (mercaptoethylamine, Sigma), DTT (1,4-Dithio-DL-Threit, Sigma), CA (Cystamine, Sigma), TCEP - (tris (2-carboxyethyl) phosphines, Sigma), DTBP (3,3'-dithio-bis-propionic acid, Fluka), J-Ac (iodoacetate, Sigma), iodoacetamide (Sigma), TMR-NHS (tetramethylrhodamine hydroxysuccinimidyl ester, Sigma).

solvent were, if necessary, Absolutely used (Fluka) or dried by standard methods. For solvent mixtures the specified mixing ratios refer to the used Volumes (v / v).

Example 7A:

Synthesis of dUYP-SS dye ( 10 ).

dUTP-AA PDTP,

20 mg dUTP-AA were dissolved in 1 ml of water and the pH was adjusted to NaOH set to 8.5. To the dUTP-AA solution was added PDTP-NHS 60mg in 0.5 ml of methanol, added dropwise with stirring. Reaction was 2 h at 40 ° C carried out. DC control: dUTP-AA-PDTP (in LM 1 Rf 0.45).

The Separation of excess PDTP-NHS and PDTP was preparative Silica gel plates, LM 2. The product, dUTP-AA-PDTP, and dUTP-AA stay on the starting line. The nucleotides were from the plate eluted with water and concentrated. This dUTP analog now has one Disulfide bond, which in a thiol exchange reaction with others Thiols can react and a fissile under mild conditions Represents connection.

Coupling of dye:

To 1ml aqueous CA solution, 200 mmol / l, pH 8.5 adjusted with NaOH, Cy3-NHS was added until the concentration of the Cy3 dye was 10 mmol / l. The reaction was stirred at RT for 10 min. Subsequently was 1 ml of TCEP solution (0.5 mol / L), pH 8.0 adjusted with NaOH, added and more Stirred for 10 min at RT. The product (Cy3-MEA) was separated on RP-18 (water-methanol gradient).

To 100μl, 10mmol / l MEA-Cy3 in 50mM borate, pH 9.5, was 50μl 30mmol / l dUTP-AA-PDTP in 50mM borate, pH 9.5 added. After 1 hour, the dUTP-SS-Cy3 was purified by thin layer chromatography, LM 1, Rf. 0.6, separated from MEA-Cy3, Rf. 0.9. Subsequently was dUTP-SS-Cy3 separated on RP-18 HPLC.

Instead of Cy3-NHS may contain another dye, e.g. TMR-NHS, Alexa 568-NHS, Alexa 590-NHS, Alexa-555-NHS in similar Be paired way.

cleavage the disulfide bond can be made by 100 mmol / l TCEP-NaOH solution.

Example 7B:

Synthesis of dUTP-SOS dye ( 11 ).

dUTP-AA-propionate-SH,

To 200μl 40mmol / l solution of dUTP-AA-PDTP was added 1ml TCEP solution 250mmol / l, pH 8, with NaOH, added and stirred for 10 min at RT.

The Separation of the nucleotides from other reagents was preparative Silica gel plates, LM 2nd product, dUTP-AA-propionate-SH, remains on the starting line. The nucleotides were eluted from the plate with water and concentrated.

This dUTP analog carries a reactive SH group that can be easily modified, for example by Maleimides or acetohalides such as iodoacetate.

Coupling of dye:

To 100 μl aqueous CA solution, 200 mmol / l, pH 8.5 adjusted with NaOH, Cy3-NHS was added, until the concentration of the Cy3 dye was 10 mmol / l. The reaction was stirred at RT for 10 min. Subsequently was 0.2 ml TCEP solution (0.5 mol / L), pH 8.0 adjusted with NaOH, added and more Stirred for 10 min at RT. The product (Cy3-MEA) was separated on RP-18 (water-methanol gradient), lyophilized and then in 100 μl 200 mmol / l acetate buffer pH 5.5 dissolved.

To 1 ml of 1 mol / l iodoacetate in DMF was added one equivalent of CDI. After 30 min at RT, 10 μl of this solution was added to 100 μl of Cy3-MEA in acetate buffer. After 30 minutes, NH ₄ HCO _{3 was added} to a concentration of 100 mmol / L and after a further 30 minutes the product, modified Cy3 dye, was purified to RP-18 in water-methanol gradient. The Cy3 dye thus obtained carries a linker containing a thioester bond and a thiol-reactive iodoacetate bond.

The resulting Cy3 dye was coupled to the dUTP-AA-propionate-SH:
To 30 μl of 5 mmol / l Cy3 derivative in 50 mmol / l borate buffer pH 9.0, 20 μl 20 mmol / l dUTP-AA-propionate-SH in 50 mmol / l borate buffer pH 9.0 were added. Reaction proceeded for 20 h at RT. Subsequently, the nucleotide was purified with the dye:
Thin-layer chromatography, LM 1, Rf. 0.6., Elution, then RP-18 column in water-methanol gradient.

Instead of Cy3-NHS may contain another dye, e.g. TMR-NHS, Alexa 568-NHS, Alexa 590-NHS, Alexa-555-NHS in similar Be paired way.

Cleavage of the thioester can be carried out by means of 100 mmol / l mercaptoethanol solution in 50 mmol / l Tris-HCl 50 mmol / l, pH 9.0, NaCl and 1 mmol / l MgCl ₂ .

Example 7C:

Synthesis of dCTP-SCO dye ( 12 ).

dCTP-PA-propionate-SH,

To 200μl 40mmol / l solution of dCTP-PA-PDTP (synthesized analogously to dUTP-AA-PDTP) 1ml TCEP solution was 250mmol / L, pH 8, adjusted with NaOH, added and stirred for 10 min at RT.

The Separation of the nucleotides from other reagents was preparative Silica gel plates, LM 2. product, dCTP-PA-propionate-SH, remains the starting line. The nucleotides were eluted from the plate with water and concentrated.

This dCTP analog carries a reactive SH group which can be easily modified, for example, under Training of thioesters.

To 20 μl of a 10 mmol / l solution of dCTP-PA-propionate-SH in 50 mmol / l borate buffer, 20% ethanol, pH 8.0 becomes one equivalent of TMR-NHS added (TMR-NHS is an isomer with the activated carboxy group in the 5-position). Reaction took place at RT for 3 h. Subsequently, the nucleotide was with the dye purified: thin layer chromatography, LM 1, Rf. 0.5., Elution, then RP-18 column in the water-methanol gradient.

Instead of TMR-NHS may contain another dye, e.g. Cy5-NHS, Cy3-NHS, Alexa-555-NNS, Alexa 568-NHS, Alexa 590-NHS in similar Be paired way.

Cleavage of the thioester can be carried out by means of 100 mmol / l mercaptoethanol solution in 50 mmol / l Tris-HCl 50 mmol / l, pH 9.0, NaCl and 1 mmol / l MgCl ₂ .

Example 7D:

Synthesis of dUTP COS dye ( 13 ).

The dUTP-R-COOCH ₃ was synthesized analogously to the protocol (Heike A. Held, Abhijit Roychowdhury, and Steven A. Benner, Nucleosides, Nucleotides & Nucleic Acids, Vol 22, 391-404 (2003)). The triphosphate synthesis was carried out according to T. Kovacs, L. Ötvös, Tetrahedron Letters, Vol 29, 4525-4588 (1988)).

500 mg (1.41 mmol) 5-iodo-2'-deoxyuridine were suspended at room temperature in 10 ml of anhydrous DMF under a nitrogen atmosphere and 10 min. touched. Now 160 mg (0.14 mmol) tetrakis (triphenylphosphine) palladium (0) are added to. After a further 10 minutes, 400 μl of triethylamine are added in succession, 480 mg (4.28 mmol) of pent-1-acid methyl ester and 55 mg (0.29 mmol) of copper (I) iodide. After 15 h, the reaction mixture concentrated on a rotary evaporator and the resulting red oil chromatographically purified on silica gel (dichloromethane: methanol = 20: 1).

400 mg (84%) of product are obtained. After triphosphorylation, dUTP-R-COOCH _3. Was obtained.

In similar Way you can also cytosine, adenosine and guanosine derivatives with the pent-1-insäuremethylester (Dissertation "Synthesis of base-modified nucleoside triphosphates and their enzymatic polymerization to functionalized DNA ", Oliver Thum, Bonn 2002).

Further modification takes place on dUTP-R-COOCH ₃ :

To 100 .mu.l of a 50 mmol / l solution of dUTP-R-COOCH ₃ in 50 mmol / l borate buffer, pH 9, is added 100 .mu.l of a 1 mol / l solution of mercaptoethanolamine, pH 9, and at 40.degree h stirred. The product of the reaction, the dUTP-R-CO-S-CH ₂ -CH ₂ -NH ₂ , is separated from the excess mercaptoethanolamine on DEAE-cellulose in borate buffer 10 mmol / l and washed with 0.3 mol / l NaCl of eluted the column.

This Nucleotide has a thioester group cleavable under mild conditions and may be modified at the amino group on the linker. To this Amino group can further modifications are made. For example, on they are coupled to a dye:

Synthesis of dUTP-COS-Cy3

To 100 μl of a 10 mmol / l solution of dUTP-R-CO-S-CH ₂ -CH ₂ -NH ₂ in 50 mmol / l borate buffer, pH 9, Cy3-NHS was added to a concentration of 15 mmol / l added. The reaction was carried out at RT for 30 min. Subsequently, the Cy3-modified nucleotide was purified on silica gel plate and RP-18 column, similarly as shown in other examples.

Instead of Cy3-NHS may contain another dye, e.g. TMR-NHS, Alexa 568-NHS, Alexa 590-NHS, Alexa-555-NHS in similar Be paired way.

Cleavage of the thioester can be carried out by means of 100 mmol / l mercaptoethanol solution in 50 mmol / l Tris-HCl 50 mmol / l, pH 9.0, NaCl and 1 mmol / l MgCl ₂ .

Such Analogs are compatible with the surface of the invention and can be used for sequencing methods become.

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Annex 1:

modification the polymerase

The Modification of the polymerase is preferably a covalent modification. examples for such modification constitutes alkylation on the SH group , e.g. with alpha-halogeno-acetyl derivatives, such as Iodoacetamide and its derivatives, iodoacetate and its derivatives, or with N-maleimide derivatives, further selective SH groups reagents are in "Chemistry of protein conjugation and crosslinking "Shan S. Wong 1993 CRC Press Inc. described. This modification can also be done by a fluorescent dye. Activated fluorescent dyes that react selectively with SH groups, are commercially available, e.g. from Molecular Probes Inc. In a preferred embodiment The invention provides a selective modification of the Klenow fragment Exo-minus of DNA polymerase on SH group of cysteine. That at the Cysts modified Klenow fragment exo-minus the DNA polymerase can in one embodiment place an unmodified Klenow fragment exo-minus the DNA polymerase be used in the enzymatic incorporation reaction.

Annex 2

Table 18, Oligonucleotides

• Column A - Designation the oligo
• Column B - manufacturer
• Column C - Sequence the oligo
• Manufacturer: MWG-Biotech, Germany, synthesized.
• Manufacturer 2: Thermo electron corporation GmbH, Ulm, Germany.
• in Oligo 31, the primer binding site is underlined.

Table 19 List of labeled oligonucleotides used in Test 1.3.

• Column A - Designation the oligo
• Column B - manufacturer
• Column C - Sequence the oligo
• Manufacturer 1: MWG-Biotech, Germany, synthesized.

Annex 3

Table 20

Explanatory notes to Table 20:

letters (U, C) represent detected signals of labeled nucleotides in one cycle, (A) stands for non-split signals.

If X, Y coordinates of the signals distributed on the surface coincide between different cycles, these signals are combined into a sequence, for example:
-UUC-: 180
-UUUUC-: 215

In the cleavage cycles and in the cycles in which a non-complementary nucleotide is missing signals on primer-template complexes. The Lack of signals with for the respective position specific coordinates will be dashed (-) shown.

Because of the bleaching of the fluorescence of the labeled nucleotides, some sequences lack signals in the center of the sequence, eg
-UUUC-: 115
-UUUC-: 162
-UUUC-: 135
-UUUC-: 138
Compare to correct sequence
-UUUUC-: 215

sequences with the same sequences of signals become a sequence group summarized. The Number after the colon indicates the number of sequences in one Sequence group.

The Sequence groups with the same number of signals (letters) become grouped in categories, Length 2, 3, 4 etc.

Legends for figures:

1 shows examples of unspecific binding and specific reactions on the surface: 1A Test 1.1 in Example 1, 1B Test 1.2 in Example 1.1, 1C Test 1.3 Oligo-T7-19-Cy3 in Example 1.1, 1D Test 1.4 in Example 1.1, 1E Hybridization of oligo-dA26-Cy3 to the surface-fixed oligo-dT31 in Example 5.1.2, 1F Specific incorporation of labeled nucleotides in example 6.1, excerpt from cycle 3, pos 2, middle.

2 shows examples of different densities of signals from single molecules. Experiments were carried out as in Example 6.1, whereby only the first incorporation of labeled nucleotides was detected. Resolution of the device was 300 nm.

3a shows the structure of the flow chamber (microchannel): A) top view, B) side view, C) schematic representation of individual components of the flow chamber at the microchannel. 1. cover glass, 2. parafilm mask, 3. slide, 4. microchannel

3b shows schematic representation of the flow direction in the exchange of solutions in the microchannel.

4a schematically shows the components of the detection apparatus: 1. camera, 2. optical path for fluorescence signals, 3. color divider with notch filter and band filter, 4. lens, 5. stage, 6. condenser, 7. light path for transmitted light, 8. light mirror, 9. light source for excitation light for fluorescence, 10. Light source for transmitted light, 11. Focusing optics with shutter for excitation light, 12. Focusing optics with shutter for transmitted light, 13. Fluorescence microscope, 14. Computer, 15. Screen for visualization of the data

4b shows a schematic representation of image recordings of the surface: 1. objective, 2. excitation light, 3. immersion oil, 4. coverslip (roof of the microchannel), 5. nucleic acid chains fixed on the surface, 6. microchannel with a solution, 7. slides (bottom of the microchannel), 8. focal plane of the lens. In this application, microchannel devices were used which include two glass surfaces modified for the purposes of this application: one on the side of the cover glass facing the microchannel, one on the side of the slide facing the microchannel. The signals were from the coverslip surface detected.

5 shows an image of the surface in Example 5.1.2 after the hybridization of dA26-Cy3 to the fixed dT31 amino nucleic acids. Figure A shows the entire image (1030 × 1300 pixels), which corresponds to 60 μm × 80 μm. Figure B shows a section of Figure A. Some signals from individual dye molecules are highlighted with circles. Rectangles mark bright signals that are mostly conglomerates of molecules.

6 shows examples of nonspecific binding at the surface: 6A Test 1.1 in Example 1, 6B Test 1.2 in Example 1.1, 6C Test 1.3 Oligo-T7-19-Cy3 in Example 1.1, 6D - Test 1.4 in Example 1.1.

7 shows sections of the surface recordings in Example 5.1.2. Figure A shows surface after testing nonspecific binding of labeled oligonucleotides. Panel B shows the same portion of the surface after specific hybridization of labeled oligonucleotides.

8th shows excerpts from the surface photographs in Example 5.1.4. Figure A shows the surface after testing non-specific binding of labeled oligonucleotides. Panel B shows the same portion of the surface after specific hybridization of labeled oligonucleotides.

9a shows sequencing on the surface with single molecules, s. Example 6.1. Shown is the same section of the surface, at different stages of cyclic sequencing from cycle 1 to cycle 20, example 6.1, MFC 1330 channel B, position 2, center. Order of adding reagents s. Table 15.

9b shows the position of the clipping in 9a in pos. 2 (picture of Pos 2 in cycle 3).

10 Schematic representation of a reversibly labeled dUTP analogue, s. Example 7A

11 Schematic representation of a reversibly labeled dUTP analogue, s. Example 7B

12 Schematic representation of a reversibly labeled dCTP analogue, s. Example 7C

13 Schematic representation of a reversibly labeled dUTP analogue, s. Example 7D

Claims (108)

surface a solid phase for Method for parallel analysis of a plurality of individual nucleic acid molecules with optical Means. Fixed phase for Method for parallel analysis of a plurality of individual nucleic acid molecules with optical Means, the surface the solid phase is a property of low nonspecific binding of having labeled components. The solid phase of claim 2, wherein the non-specific binding of labeled components is a binding of less than 200 events / 100 μm ² during analysis. The solid phase of claim 2, wherein the non-specific binding of labeled components is a binding of less than 100 events / 100 μm ² during analysis. The solid phase of claim 2, wherein the non-specific binding of labeled components is a binding of less than 50 events / 100 μm ² during analysis. The solid phase of claim 2, wherein the non-specific binding of labeled components is a binding of less than 20 events / 100 μm ² during analysis. The solid phase of claim 2, wherein the non-specific binding of labeled components is a binding of less than 10 events / 100 μm ² during analysis. The solid phase of claim 2, wherein the nonspecific binding of labeled components is a binding of less than 5 events / 100 μm ² during analysis. The solid phase of claim 2, wherein the non-specific binding of labeled components is a binding of less than 2 events / 100 μm ² during analysis. A solid phase according to claims 1 to 9, wherein the solid Phase a silica surface includes A solid phase according to claims 1 to 9, wherein the solid Phase a glass surface includes The solid phase of claims 1 to 9, wherein the solid phase includes an SiO ₂ surface A solid phase according to claims 1 to 9, wherein the solid Phase a Si-OH surface includes The solid phase according to claims 1 to 13, wherein the surface of the solid phase is flat Solid phase according to claims 1 to 14, wherein on the surface of this fixed phase nucleic acid chains are. A solid phase according to claims 1 to 14, wherein attached thereto solid phase nucleic acid chains via a linker are fixed. Method for the preparation of the solid phase according to claims 1 to 16, wherein the production includes the following steps: 1. Removal of the outer layer the solid phase 2. Coupling of nucleic acid chains to the solid phase Method for the preparation of the solid phase according to claims 1 to 16, wherein the production includes the following steps: 1. Removal of the outer layer the solid phase 2. Coupling of a linker layer to the solid phase 3. Coupling of nucleic acid chains to the linker layer Method for the preparation of the solid phase according to claims 1 to 16, wherein the production includes the following steps: 1. Removal of the outer layer the solid phase 2. Cyclic Synthesis of Nucleic Acid Chains at the solid phase Method for the preparation of the solid phase according to claims 1 to 16, wherein the production includes the following steps:  1. Removal of the outer layer the solid phase 2. Coupling of a linker layer to the solid phase 3. Cyclic synthesis of nucleic acid chains on the solid phase Method for the preparation of the solid phase according to claims 1 to 16, wherein the production includes the following steps: 1. Removal of the outer layer the solid phase 2. Coupling of nucleic acid chains to the solid phase Third Coupling of other substances Method for the preparation of the solid phase according to claims 1 to 16, wherein the production includes the following steps: 1. Removal of the outer layer the solid phase 2. Coupling of a linker layer to the solid phase 3. Coupling of nucleic acid chains to the linker layer 4th Coupling of other substances Method for the preparation of the solid phase according to claims 1 to 16, wherein the production includes the following steps: 1. Removal of the outer layer the solid phase 2. Cyclic Synthesis of Nucleic Acid Chains at the solid phase 3. Coupling of other substances Method for the preparation of the solid phase according to claims 1 to 16, wherein the production includes the following steps: 1. Removal of the outer layer the solid phase 2. Coupling of a linker layer to the solid phase 3. Cyclic synthesis of nucleic acid chains on the solid phase 4th Coupling of other substances Method for the preparation of the solid phase according to claims 1 to 24, the outer layer only partially removed. Method for the preparation of the solid phase according to claims 1 to 25, the removal of the outer layer by chemical reaction Method for producing the solid phase according to claim 26, wherein the chemical reaction takes place in the presence of HF Method for producing the solid phase according to claim 26, wherein the chemical reaction takes place in the presence of NaOH Method for the preparation of the solid phase according to claims 1 to 28, wherein the thickness of the removed outer layer between 1 nm and 10 nm. Method for the preparation of the solid phase according to claims 1 to 28, wherein the thickness of the removed outer layer between 10 nm and 100 nm. Method for the preparation of the solid phase according to claims 1 to 28, wherein the thickness of the removed outer layer between 100 nm and 1 μm lies. Method for the preparation of the solid phase according to claims 1 to 28, wherein the thickness of the removed outer layer is between 1 .mu.m and 10 .mu.m. Method for the preparation of the solid phase according to claims 1 to 28, wherein the thickness of the removed outer layer is between 10 .mu.m and 100 .mu.m. Method for the preparation of the solid phase according to claims 1 to 28, wherein after the removal of the outer layer of the solid phase no drying steps take place and further steps take place in the liquid phase. Method for producing the solid phase according to claims 21 to 24, where further substances include the following substances: monomers (Phosphate, sulphate, carboxy groups), polymers (polyethyleneglycol, polyacrylates, polyacrylamides, polyphosphates) Fixed phase for Method for parallel analysis of a plurality of individual nucleic acid molecules with optical Agents prepared by methods in claims 17 to 35 becomes. Fixed phase for Method for parallel analysis of a plurality of individual nucleic acid molecules with optical Agents prepared by methods in claims 17 to 35 being, being at the surface nucleic acid chains are fixed. Solid phase according to claims 1 to 37 with fixed Nucleic acid chains, wherein fixed nucleic acid chains represent a uniform population Solid phase according to claims 1 to 37 with fixed Nucleic acid chains, wherein fixed nucleic acid chains represent several different populations Solid phase according to claims 1 to 39 with fixed Nucleic acid chains, wherein nucleic acid chains a Length between Have 5 and 50 nucleotides Solid phase according to claims 1 to 39 with fixed nucleic acid chains, wherein nucleic acid chains have a length between 20 and 200 nucleotides Solid phase according to claims 1 to 39 with fixed Nucleic acid chains, wherein nucleic acid chains a Length between Have 50 and 500 nucleotides Solid phase with fixed nucleic acid chains according to claims 1 to 42, wherein the fixation of nucleic acid chains is covalent. Solid phase with fixed nucleic acid chains according to claims 1 to 42, wherein the fixation is affine. Solid phase according to claims 16, 18, 20, 22, 24, wherein the linker is a length from 1 to 50 nm Solid phase according to claims 16, 18, 20, 22, 24, 45, wherein the linker is a non-branched polymer Solid phase according to claims 16, 18, 20, 22, 24, 45 wherein the linker is a branched polymer Solid phase according to claims 16, 18, 20, 22, 24, wherein Streptavidin or avidin acts as a linker. Solid phase according to claims 16, 18, 20, 22, 24, wherein nucleic acid chains occur as a linker. Solid phase with fixed nucleic acid chains according to claims 1 to 49, wherein the density of the fixed nucleic acid chains is between 1/100 μm ² and 10/100 μm ² . Solid phase with fixed nucleic acid chains according to claims 1 to 49, wherein the density of the fixed nucleic acid chains is between 10/100 μm ² and 100/100 μm ² . Solid phase with fixed nucleic acid chains according to claims 1 to 49, wherein the density of the fixed nucleic acid chains is between 100/100 μm ² and 1000/100 μm ² . Solid phase with fixed nucleic acid chains according to claims 1 to 49, wherein the density of the fixed nucleic acid chains is between 1000/100 μm ² and 10,000 / 100 μm ² . The fixed phase with fixed nucleic acid chains according to claims 1 to 49, wherein the density of the fixed nucleic acid chains is between 10,000 / 100 μm ² and 1,000,000 / 100 μm ² . Solid phase with fixed nucleic acid chains according to claims 1 to 54, wherein one recognizable by optical means on the solid phase Pattern is fixed Solid phase with fixed nucleic acid chains according to claims 1 to 55, wherein the solid phase is part of a device for replacement of liquids represents. Solid phase with fixed nucleic acid chains according to claims 1 to 56, wherein the solid phase for electromagnetic radiation with wavelengths in the range between 200 nm to 400 nm is permeable. Solid phase with fixed nucleic acid chains according to claims 1 to 56, wherein the solid phase for electromagnetic radiation with wavelengths in the range between 200 nm to 2000 nm is permeable. Solid phase with fixed nucleic acid chains according to claims 1 to 56, wherein the solid phase for electromagnetic radiation with wavelengths in the range between 400 nm to 800 nm is permeable. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Agents, wherein the solid phase is used according to claims 1 to 59. Method for the parallel analysis of a multitude single nucleic acid molecules with optical The agent of claim 60, wherein labeled components are used become. Method for the parallel analysis of a multitude single nucleic acid molecules with optical The agent of claim 61, wherein the label is from the labeled Components can be removed by cleavage. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 61 and 62, wherein labeled components labeled ribonucleoside triphosphates or deoxyribonucleoside triphosphates or dideoxyribonucleoside triphosphates lock in. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 61 to 63, wherein labeled components reversibly labeled Ribonukleosidtriphosphate or Deoxyribonucleoside triphosphates or dideoxyribonucleoside triphosphates lock in. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 61 and 62, wherein labeled components include labeled nucleic acids. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claim 65, wherein labeled components are reversible labeled nucleic acids lock in. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60 to 66, wherein in parallel 100 to 10,000 nucleic acid chains to be analyzed Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60 to 66, wherein in parallel 1000 to 100,000 nucleic acid chains to be analyzed Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60 to 66, wherein in parallel 10,000 to 1,000,000 nucleic acid chains to be analyzed Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60 to 66, wherein in parallel 100,000 to 10,000,000 nucleic acid chains to be analyzed Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60 to 66, with parallel 1,000,000 to 100,000,000 nucleic acid chains to be analyzed Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60 to 66, wherein in parallel 100,000,000 to 10,000,000,000 nucleic acid chains to be analyzed Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60 to 72, wherein events on individual nucleic acid chain molecules optically be detected Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60 to 73, wherein optical means allow a resolution of up to 0.3 microns Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60 to 73, wherein optical means allow a resolution up to 0.5 microns Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60 to 73, wherein optical means allow a resolution up to 0.8 microns Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60 to 73, wherein optical means allow a resolution of up to 1 micron Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60 to 73, wherein optical means allow a resolution of up to 1.2 microns Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60 to 73, wherein optical means allow a resolution of up to 1.5 microns Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60 to 73, wherein optical means allow a resolution of up to 2 microns Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60-80, the analysis being by cyclic sequencing. Method for the parallel analysis of a multitude single nucleic acid molecules with optical The agent of claim 81, wherein the analysis is by cyclic sequencing extends and includes the following steps: 1. Provision of a solid phase according to claims 1 to 59 2. binding of nucleic acid molecules to be analyzed thereon solid phase to form extensible primer-template complexes, Third execution a cyclic reaction with those fixed on the solid phase Nucleic acid chains, includes the following steps: 3.1 Incubation of a solution with one or more polymerase and one or more labeled Nucleotides (polymerase-nucleotide mixture) with the solid phase below Conditions involving an incorporation reaction on the formed primer-template complexes allow, 3.2 Removal of the polymerase-nucleotide mixture and washing the solid phase 3.3 Detection of the signals from incorporated labeled nucleotides, where relative coordinates are individual Signals are identified 3.4 Removal of signals from built-in nucleotides, 3.5 optionally washing the solid phase 3.6 if necessary repeat steps 3.1 to 3.5 4. Reconstruction of the Sequences of Individual Nucleic Acid Molecules the signals obtained in step 3.3 Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60 to 82, wherein the nucleic acid molecules to be analyzed nucleic acid chains with a length between Represent 30 and 300 nucleotides. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60 to 82, wherein the nucleic acid molecules to be analyzed nucleic acid chains with a length between Represent 30 and 3000 nucleotides. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60 to 84, wherein the nucleic acid molecules to be analyzed Deoxyribonukleinsäureketten or ribonucleic acid represent. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60 to 85, wherein the provided solid phase carries fixed nucleic acids, the as a primer for to serve the sequencing. Method for the parallel analysis of a multitude single nucleic acid molecules with optical The agent of claim 86, wherein the provided solid phase fixed nucleic acids wearing, as primers for the sequencing serve and the nucleic acid molecules to be analyzed directly hybridized to these primers, with extension-capable primer-template complexes be formed. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60 to 85, wherein the nucleic acid molecules to be analyzed fixed to the solid phase and then a primer is hybridized to the nucleic acid chains to be analyzed. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60 to 82, wherein the fixation of nucleic acid molecules to be analyzed is covalent takes place on the solid phase. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60 to 82, wherein the fixation of nucleic acid molecules to be analyzed affin takes place on the solid phase. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 82 and 90, wherein the provided solid phase is fixed nucleic acid chains wearing, as an anchor for serve affine fixation of nucleic acid molecules to be analyzed. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 82 and 90, wherein the fixation of nucleic acid chains to be analyzed by the hybridization or affine binding to the anchor at the solid phase takes place. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60 to 92, wherein the density of the fixed extensible template-template complexes on the solid phase is between 1 and 10 complexes per 100 μm 2. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60 to 92, wherein the density of the fixed extensible template-template complexes on the solid phase is between 1 and 100 complexes per 100 μm2. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60 to 94, wherein the nucleic acid molecules to be analyzed on the solid phase in a random arrangement be fixed Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 60 to 94, wherein the nucleic acid molecules to be analyzed to the solid phase in a predetermined Arrangement to be fixed Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 82 to 96, wherein nucleotides used in step 3.1 ribonucleoside triphosphates or deoxyribonucleoside triphosphates or dideoxyribonucleoside triphosphates lock in. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 82 to 97, wherein used in step 3.1 nucleotides one reversible carry terminating group, leaving only one labeled nucleotide can be installed in an incubation 3.1 Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claim 98, wherein after the detection step 3.3 a another step to remove the reversibly terminating group im done so that the extension reaction takes place in the further course can Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 82 to 99, nucleotides used being a uniform label carry and in the incubation 3.1 modified nucleotides same Base type can be added. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 82 to 99, wherein nucleotides used a base-specific label carry and in the incubation 3.1 modified nucleotides different Base type can be added. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 82 to 101, wherein in step 3.3 relative intensity of a each signal is determined. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 82 to 101, where the number of repetitions of steps 3.1 to 3.5 is between 2 and 10. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 82 to 101, where the number of repetitions of steps 3.1 to 3.5 is between 10 and 25. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 82 to 101, where the number of repetitions of steps 3.1 to 3.5 is between 25 and 50. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claims 82 to 101, where the number of repetitions of steps 3.1 to 3.5 is between 50 and 200. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Compositions according to claims 60 to 80, wherein the analysis is by hybridization complementary labeled nucleic acid chains runs. A method of analyzing nucleic acid chains with the solid phase of claims 1 to 59, wherein the nucleic acid chains to be analyzed are fixed to the solid phase and the analysis is carried out by the hybridization of complementary labeled nucleic acid chains.