DE102004025746A1 - Parallel sequencing of nucleic acids by optical methods, by cyclic primer-matrix extension, using a solid phase with reduced non-specific binding of labeled components - Google Patents

Abstract

Method for parallel sequence analysis of nucleic acids (NA) by optical means. Method for parallel sequence analysis of nucleic acids (NA) by optical means comprises: (A) preparing a solid phase (SP); (B) binding NA being analyzed to SP, with formation of extensible primer-matrix complexes (X); (C) performing cyclic reactions on (X) and (D) reconstructing the sequences of individual NA from the signals generated in (c). Step (c) comprises (i) enzymatic incorporation of a labeled nucleotide (nt) into (X); (ii) identifying the incorporated nt by determining its relative coordinates; (iii) removing the signal of the labeled nt; (iv) washing SP and (v) repeating steps (i)-(iv). Independent claims are also included for the following: (1) SP for the new process where the surface has reduced non-specific binding of labeled components; (2) method for preparing SP; (3) SP prepared by method (2); and (4) method for parallel analysis of many NA using SP.

Description

Description of the invention

1. Introduction

1.1 classification in the technological field

The The invention relates to a method for analyzing nucleic acid chains. The basis of the method is the detection of fluorescence signals individual labeled with dye nucleotide molecules, the be incorporated by a polymerase in a growing nucleic acid chain. The reaction is ongoing on a flat surface. To this surface Many single nucleic acid molecules are bound. All these nucleic acid molecules are exposed to the same conditions, so that all nucleic acid molecules simultaneously a build-up reaction can take place.

• 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 enzymatischer Einbau von markierten Nukleotiden an den 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

The method for the parallel sequence analysis of nucleic acid sequences (nucleic acid chains, NSKs) with optical means, which 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 enzymatic incorporation of labeled nucleotides on 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

Nucleotide molecules are marked with one or more fluorescent markers. The fluorescent markers can both low molecular weight and macromolecular.

One Another object of the invention is a surface of a solid phase for the method for sequencing nucleic acid chains. Because the surface is a critical role in the process, the surface is first described.

The Nucleic acid chain sequence analysis is in many areas of science, medicine and industry become an important tool. For analysis, several Developed process.

The Best known methods are chain termination sequencing According to Sanger (F. Sanger et al PNAS 1977 v.74 p 5463), which was published on the Incorporation of chain terminators, and the Maxam-Gilbert method, the base-specific modification and cleavage of nucleic acid chains (A.M. Maxam and W. Gilbert PNAS 1977, v.74 p.560). Both Methods provide a number of different nucleic acid chain fragments Lengths. These fragments become the length after being separated in a gel. In this case, all the disadvantages of electrophoresis (such as long term, relatively short stretches of sequences, which can be determined in one approach, limited number of parallel approaches as well as relatively large Amounts of DNA) can be accepted. These methods are very labor intensive and slow.

One another sequencing method based on hybridization of nucleic acid chains with short oligonucleotides. This is done using mathematical methods Calculates how many oligonucleotides of a given length exist have to be to determine a complete sequence (Z.T. Strezoska et al., PNAS 1991 v.88 p.10089, R. S. Drmanac et al. Science 1993 v.260 p.1649). This process also has problems: it can only be one Sequence can be determined in one approach, secondary structures disrupt the Hybridization and repeats prevent the correct one Analysis.

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 fixed in a solid phase and subsequently in a biochemical reaction, for example, hybridization or primer extension 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.

1.3 Terms / Definitions:

1.3.1. Macromolecular connection

One Molecule, a molecular complex, a nanocrystal or nanoparticle whose mass is between 2kDa and 100kDa, 100kDa and 200kDa, 200kDa and 1000kDa or 1MDa and 100MDa or 100 MDa and 1000Da. Examples of macromolecular compounds are nucleic acids, like oligonucleotides with a length of more than 10 nucleotides, polynucleotides, polypeptides, proteins or Enzymes, quantum dots, polymers such as PEG, Mowiol, polyacrylate, nanogold particles.

1.3.2. low molecular weight connection

One molecule or a molecular complex, whose mass is less than 2000 Da (2kDa), e.g. natural nucleotides, dATP, dUTP, many dyes such as Cy3, rhodamine, fluorescein, linker with an average length between 5 and 30 atoms, rare earths and conventionally modified Nucleotides, such as biotin-16-dUTP.

1.3.3. A nuc macromolecule

Nuk

– eine Nuk-Komponente ist

Linker

– eine Linker-Komponente ist

Marker

– eine Makrer-Komponente ist

n

– eine ganze Zahl zwischen 1 und 100 ist

For the purposes of this application, a chemical structure which has one or more nuc-components, one or more linker components and a marker component, 15 or 16 :
(Nuk linker) _n marker
in which:

Nuk

- is a nuc component

left

- is a linker component

marker

- is a Makrer component

n

- is an integer between 1 and 100

Nuk

– ein Nukleotid oder ein Nukleosid ist (Nuk-Komponente)

L

– ein Teil des Linkers ist, das die Verbindung zwischen Nuk und dem Linkerrest darstellt (Kopplungseinheit L)

T

– ein Teil des Linkers ist, das die Verbindung zwischen dem Linkerrest und dem Marker darstellt (Kopplungseinheit T)

Polymer

– ein Teil des Linkers ist, das ein wasserlösliches Polymer mit einer durchschnittlichen Länge zwischen 100 und 10.000 Atome ist (Kopplungseinheit L, Polymerlinker, Kopplungseinheit T sind bei dieser Ausführungsform als Linker-Komponente zusammengefaßt)

Marker

– eine Marker-Komponente

n

– eine ganze Zahl zwischen 1 und 100

In a preferred embodiment, the linker component consists of a coupling unit L for the coupling of the linker to the nuc-component, of a water-soluble polymer and of a coupling unit T for the coupling of the linker to the marker component. In this preferred embodiment, a nuc-macromolecule has the following structure, 15 or 16 :
(Nuk-L polymer T) _n label
in which:

Nuk

Is a nucleotide or a nucleoside (nuc-component)

L

Is a part of the linker which represents the connection between nuc and the linker residue (coupling unit L)

T

Is a part of the linker that represents the link between the linker residue and the marker (coupling unit T)

polymer

Is a part of the linker which is a water-soluble polymer having an average length between 100 and 10,000 atoms (coupling unit L, polymer linker, coupling unit T are combined in this embodiment as a linker component)

marker

- a marker component

n

- an integer between 1 and 100

Nuc-macromolecules are by a combination of one or more nuc-components, accordingly one or more long linker components and a marker component Are defined.

1.3.3.1 Nuc component

Nuk component can be both a nucleotide and a nucleoside. In the following, nucleotides will be considered in the description. It should be obvious to a person skilled in the art that nucleosides can also be modified in a corresponding manner and be converted into corresponding reactions. In principle, all conventional nucleotide variants suitable as substrate for nucleotide-accepting enzymes can serve as nuc-component of the nuc-macromolecule, so that both natural and modified nucleotides (nucleotide analogues) are suitable for the nuc-component. For modified nucleotides, base, sugar or phosphate moieties can be modified, 17A and B. Many examples of nucleotide modifications are known to those skilled in the art ("Advanced Organic Chemistry of Nucleic Acids", 1994, Shabarova, ISBN 3-527-29021-4, "Nucleotide Analogs" Scheit, 1980, ISBN 0-471-04854 -2, "Nucleosides and Nucleic Acid Chemistry", Kisakürek 2000, "Anti-HIV Nucleosides" Mitsuya, 1997, "Nucleoside Analogs in Cancer Therapy", Cheson, 1997) in the text, other examples of the modifications of the nucleotides are also given.

The Nuc component preferably has a base component (base), a Sugar component (sugar) and a phosphate component (phosphate).

1.3.3.1.1 Variations on the phosphate

In an embodiment the nuc-component is a nucleoside. In another embodiment the nuc-component is a nucleoside monophosphate. In one another embodiment the nuc-component is a nucleoside diphosphate. In another embodiment the nuc-component is a nucleoside triphosphate. Also higher phosphate derivatives (Tetraphosphate, etc.) can be used.

The mentioned phosphate modifications can as natural Nucleoside triphosphates at the 5'-position or at other positions of the sugar part of the nucleotide sit, for example at 3 'position.

1.3.3.1.2 Variations at the base

The Nuk component can be a naturally occurring in the nucleic acids Nucleotide (with one of the bases adenine, guanine, thymine, Cytosine, uracil, inosine), or a modified base, e.g. 7-deazaadenine, 6-thio-adenine, included, literature s. above..

1.3.3.1.3 Variations on the sugar

Both ribose and 2'-deoxyribose or 2 ', 3'-dideoxyribose may be the backbone of the Nuk components form. It is also possible to use 3'-hydroxyl-modified ribose or 2'-deoxyribose. The application refers to 2'-deoxynucleotides, for example 2'-deoxyuridine triphosphate, 2'-deoxycytidine triphosphate, 2'-deoxyadenosine triphosphate, 2'-deoxyguanosine triphosphate as dUTP, dCTP, dATP and dGTP.

1.3.3.1.4 Coupling of NT and linker

The Nuk component is connected to the linker at a coupling site. The coupling site of the linker to the nuc-component may be at the base, on the sugar (ribose or deoxyribose), or on the phosphate part are located.

The Connection between the linker component and the nuk component is preferably covalent.

If the coupling site is at the base, it is preferably at positions 4 or 5 at pyrimidine bases and at positions 6, 7, 8 at the purine bases. At the sugar can the positions 2 ', 3 ', 4' or 5 'the coupling points represent. The coupling to the phosphate groups can occur at the alpha, beta, or gamma phosphate group. Examples of the coupling site at the base are in Short WO 9949082, Balasubramanian WO 03048387, Tcherkassov WO 02088382 (see also commercially available Nucleotides (Amersham, Roche), at the ribose in Herrlein et al. Helvetica Chimica Acta, 1994, V. 77, p. 586, Jameson et al. Method in Enzymology, 1997, V. 278, p. 363, Canard US. Pat. 5798210, Kwiatkowski US Pat. 6255475, Kwiatkowski WO 01/25247, Parce WO 0050642., to phosphate groups in Jameson et al. Method in Enzymalogy, 1997, V. 278, p 363 described.

The Position of the coupling point hangs from the field of application of NT macromolecules. For example for the Labeling of nucleic acids, at the nucleic acid strand should remain, preferably coupling sites on the sugar or on the base used. The coupling to the gamma or beta phosphate groups can be done, for example, when the mark during installation of the nuc macromolecule shall be.

The Connection between the nuc-component and the linker component over a coupling unit (L) which is a part of the linker component.

The Connection between the nuc-component and the linker can be in one embodiment resistant be, e.g. at temperatures up to 130 ° C, for pH ranges between 1 and 14, and / or resistant to hydrolytic enzymes (e.g., proteases, esterases) be. In another embodiment the invention is the connection between the nuc-component and the linker under mild conditions fissile.

These fissile connection allows the removal of linker and marker components. This can be, for example in the methods of sequencing by the synthesis of importance such as Pyrosequencing, BASS (Canard et al., U.S. Patent 5,798,210, Rasolonjatovo nucleosides & nucleotides 1999, V.18 5.1021, Metzker et al. NAR 1994, V.22, p.4259, Welch et al. Nucleosides & nucleotides 1999, V.18, p.19) or single molecule sequencing, Tcherkassov WO 02088382. Your choice is not limited, unless under the conditions the enzymatic reaction remains stable, no irreversible disruption of Enzymes (e.g., polymerase) and under mild conditions can be split off. Under "mild Conditions "are to understand such conditions, for example, nucleic acid primer complexes do not destroy, whereby e.g. the pH preferably between 3 and 11 and the temperature between 0 ° C and a temperature value (x). This temperature value (x) depends on the Tm of the nucleic acid primer complex (Tm is the melting point) and is used, for example, as Tm (nucleic acid-primer complex) minus 5 ° C calculated (e.g., Tm is 47 ° C, then the maximum temperature is 42 ° C; under these conditions Ester, thioester, acetals, phosphoester, Disulfide compounds and photolabile compounds as cleavable compounds).

Preferably, said cleavable compound belongs to chemically or enzymatically cleavable or photolabile compounds. As examples of chemically cleavable groups, ester, thioester, disulfide, acetal compounds are preferred (Short WO9949082, "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., Biol., 1976, V. 104 243, "Chemistry of carboxylic acids and esters." S. Patai 1969 Interscience Publ.). Examples of photolabile compounds can be found in the following references Rothschild WO 9531429, "Protective Groups in Organic Synthesis" 1991 John Wiley & Sons, Inc., V. Pillai Synthesis 1980, p. V. Pillai Org. Photochem. 1987 V.9 p.225, Dissertation "New Photolabile Protecting Groups for Light-Controlled Oligonucleotide Synthesis" H.Giegrich, 1996, Konstanz, Dissertation "New Photolabile Protecting Groups for Light-Controlled Oligonucleotide Synthesis" SMBühler, 1999, Konstanz).

1.3.3.1.5 Number of linked Nuc-components

In an embodiment According to the invention, only one nuc-component is coupled per one nuc-macromolecule. In another embodiment According to the invention, several nuc-components are coupled per nuc-macromolecule. Several nuc components can be uniform or different, for example, average 2 to 5, 5 to 10, 10 to 25, 25 to 50, 50 to 100, 100 to 250, 250 to 500, 500 to 1000 nuc-components per one nuk macromolecule coupled could be.

1.3.3.2 Linker component

The Designation Linker or linker component becomes synonymous in the application used and refers to the entire structural section of the nuc macromolecule between the nuk component and the marker component.

1.3.3.2.1 Linker components

• 1) Kopplungseinheit L
• 2) wasserlösliches Polymer
• 3) Kopplungseinheit T

The linker component is part of the nuclomor molecule that lies between the respective nuc-component and the marker component. The linker component preferably has the following components in its structure:

• 1) coupling unit L
• 2) water-soluble polymer
• 3) coupling unit T

The Division of the linker component into individual components is pure functional and intended only to illustrate the structure serve. Depending on the perspective, individual structures of the Linkers calculated on the one hand or on the other functional component become.

The coupling unit L has the function of connecting the linker component and the nuk component. Their structure is not limited as long as the nuc macromolecule is accepted as a substrate by the enzymes. Preference is given to short, unbranched compounds, up to max. 20 atoms in length. The particular structure of the coupling unit L depends on the coupling site of the linker to the nucleotide and of the respective polymer of the linker. Some examples of the coupling units L are given in Examples 1 to 33. Many conventionally modified nucleotides carry a short linker, these short linkers serve as further examples of the coupling unit L, eg short linkers on the base: Short WO 9949082, Balasubramanian WO 03048387, Tcherkassov WO 02088382 (see also commercially available nucleotides (Amersham, Roche) Helvetica Chimica Acta, 1994, V. 77, p 586, Jameson et al., Method in Enzymology, 1997, V. 278, p 363, Canard US Pat. Kwiatkowski US Pat. No. 6255475, Kwiatkowski WO 01/25247, Parce WO 0050642., Short Linker to Phosphate Groups in Jameson et al., Method in Enzymology, 1997, V. 278, p 363 Still further examples of the coupling unit L are given below:
R ₆ -NH-R ₇ , R ₆ -OR ₇ , R ₆ -SR ₇ , R ₆ -SS-R ₇ , R ₆ -CO-NH-R ₇ , R ₆ -NH-CO-R ₇ , R ₆ -CO-OR ₇ , R ₆ -O-CO-R ₇ , R ₆ -CO-SR ₇ , R ₆ -S-CO-R ₇ , R ₆ -P (O) ₂ -R ₇ , R ₆ -Si R ₇ , R ₆ - (CH ₂ ) _n -R ₇ , R ₆ - (CH ₂ ) _n -R ₇ , R ₆ -A- (CH ₂ ) _n -R ₇ , R ₆ - (CN ₂ ) _n -BR ₇ , R ₆ - (CH = CH-) _n -R ₇ , R ₆ - (A-CH = CH-) _n -R ₇ , R ₆ - (CH = CH-B-) _n -R ₇ , R ₆ -A-CH = CH- (CH ₂ -) _n -R ₇ , R ₆ - (- CH = CH-CH ₂ ) _n -BR ₇ , R ₆ - (- CH = CH-CH ₂ -CH ₂ ) _n -BR ₇ , R ₆ - (C≡C-) _n -R ₇ , R ₆ - (AC≡C-) _n -R ₇ , R ₆ - (C≡CB-) _n -R ₇ , R ₆ -AC≡C- (CH ₂ ) _n -R ₇ , R ₆ - (- C≡C-CH ₂ ) _n -BR ₇ , R ₆ - (- C≡C-CH ₂ -CH ₂ ) _n -BR ₇ .
wherein R _{6 is} the nuc-component, R _{7 is the} polymer and A and B include the following structural elements: -NH-, -O-, -S-, -SS-, -CO-NH-, -NH- CO, -CO-O-, -O-CO-, -CO-S-, -S-CO-, -P (O) ₂ -, -Si-, - (CH ₂ ) _n -, a photolabile group where n - is 1 to 5

The Coupling unit L is on a side with the Nuk component, on the other covalently bonded to polymer. The type of coupling depends on the type of polymer. In a preferred form, the polymer has reactive groups, for example NH 2 (amino), OH (hydroxy), SH (mercapto), COOH (carboxy), CHO (aldehyde), acrylic or maleimide, Halogen groups. Such polymers are commercially available (e.g., Fluka). Some variants for the coupling of polymers to the coupling units are under Examples 1 to 33 indicated.

The water-soluble Polymer forms the major portion in a preferred embodiment the linker component. It is a polymer, preferably hydrophilic, consisting of the same or different monomers. Examples for suitable Polymers make polyethylene glycol (PEG), polysaccharides, polyamides (e.g. Polypeptides), polyphosphates, polyacetates, poly (alkylene glycols), Copolymers of ethylene glycol and propylene glycol, poly (olefinic Alcohols), poly (vinylpyrrolidones), poly (hydroxyalkylmethacrylamides), Poly (hydroxyalkyl methacrylates), poly (x-hydroxy acids), poly-acrylic acid and their derivatives, poly-acrylamides in their derivatives, poly (vinyl alcohol), Polylactate acid, polyglycolic acid, Poly (epsilon-caprolactones), Poly (beta-hydroxybutyrate), poly (beta-hydroxyvalerate), polydioxanone, Poly (ethylene terephthalate), poly (malic acid), poly (tartronic acid), poly (ortho esters), polyanhydrides, polycyanoacrylates, poly (phosphoesters), polyphosphazenes, Hyaluronidates, polysulfones.

This Polymer may or may not be branched. This polymer can be made consist of several sections of different lengths, each one Section consists of the same monomers and monomers in different Sections are different. A specialist should be obvious appear that for a macromolecular linkers usually only an average mass can be determined so that the information to the molecular weights ("macromolecules, chemical structure and Syntheses ", volume 1, 4, H. Elias, 1999, ISBN 3-527-29872-X). That's why for nuc macromolecules no exact Mass be made.

1.3.3.2.2 Linker length

The linker length is between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 10,000, 10,000 to 100,000 atoms, thus the average Linker length between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 10,000, 10,000 to 100,000 angstroms (measured on a potentially maximally stretched molecule).

Some Sections of the linker can contain rigid areas and other sections can be flexible areas contain.

It will be apparent to those skilled in the obvious that the listed linker lengths a considerably larger molecule size and mass can have, as the respective nuc-component itself.

1.3.3.2.3 Linker coupling in a nuc macromolecule

Of the Linker is on one side to the nuk component and on the other Bound to the marker component. To do this, the linker has its end coupling units that perform this function. The connection with the nuk components was discussed above. The link between the linker and the marker components is via coupling unit T. Their structure is not limited as long as the nuc macromolecule as a substrate is accepted by the enzymes. Preferred are short, unbranched Connections, up to max. 20 atoms in length. The respective structure the coupling unit T hangs from the coupling site to the marker component and from the respective one Polymer of the linker. The coupling unit T is with the polymer covalently linked. The type of coupling depends on the type of polymer from. In preferred form, the polymer has reactive ends Groups, for example NH 2 (amino), OH (hydroxy), SH (mercapto), COOH (carboxy), CHO (aldehyde), acrylic or maleimide, halogen groups. Such polymers are commercially available (e.g., Fluka). Some examples of the coupling units L are given in Examples 1 to 33, Further examples of the chemical and affine couplings s. in the literature: "Chemistry of protein conjugation and crosslinking "Shan S. Wong 1993," Bioconjugation: protein coupling techniques for the biomedical sciences ", M. Aslam, 1996.

Of the Linker may also contain other functional groups or sections, for example one or more fissile under mild conditions Groups.

A fissile connection within the linker allows removal of one Part of the linker and the marker component. After the split remains a linker residue on the nuc-component, examples of cleavable Connections are given in section 1.3.3.1.4.

1.3.3.3 Marker component

The marker component can have different structures. The structures in detail are not limited as long as they do not complete the substrate properties of the nuc-components for enzymes cancel. This structure preferably has a signaling or a signal-switching function. The marker may also have other functions, such as structural, antitoxic, or affine function (for example, in drugs or transmissions).

1.3.3.3.1 The composition the marker component

Of the Marker can be made from a macromolecule consist of a group of the same or different structural Units (marker units) with signaling or signal-transmitting Function. These units can molecules low molecular weight, e.g. under 2000 Da, or even yourself Be macromolecules. The number of signaling or signal-transmitting is Units that are grouped together to form a marker component, between 1 and 2, 2 and 5, 5 and 20, 20 and 50, 50 and 100, 100 and 500, 500 and 1000. The units are macromolecular framework bound, the core components of the marker. This core component connects the units to each other and establishes the connection to one or more nuc-linker components.

1.3.3.3.2 Structure of the signaling or the signal-transmitting units of the marker

The structural marker units can selected from the following groups become:

1.3.3.3.2.1 Structures with low molecular weight:

Biotin molecules, hapten molecules (eg digoxigenin), radioactive isotopes (eg P ³² , J ¹³¹ ), or their compounds, rare earths, dyes, fluorescent dyes (eg Molecular Probes, Inc.) with the same or different spectral properties, dyes, which are FRET among each other.

Also chemically reactive groups, such as amino, mercapto, Aldehyde, iodoacetate, acrylic, dithio, thioester compounds can be used as serve signal-transmitting structural units. After installation can these reactive groups with signaling elements, such as Dyes with corresponding reactive groups (for example NHS esters, mercapto, amino groups). The basics for the Choosing a suitable pair are described in "Chemistry of protein conjugation and crosslinking "Shan S. Wong 1993.

In a special embodiment He feels the combination of a markomolecular linker component and Already meets the requirements of a low molecular weight marker present invention. Such compounds are also subject matter of this invention, since they are used both as intermediates for the chemical Synthesis of nuc macromolecules with a macromolecular marker, e.g. dUTP-PEG-biotin, as well independent can be used in the enzymatic reactions, such as for example, with only one dye labeled nucleotides. When Dyes can different fluorescent dyes occur, their choice is not limited, as long as they do not significantly affect the enzymatic reaction. Examples for dyes Rhodamine (Rhodamine 110, tetramethylrhodamine), cyanine dyes (Cy2, Cy3, Cy5, Cy7), Coumarins, Bodipy, Fluorescein, Alexa Dyes. Many dyes are commercially available, for example from Molecular Probes Europe, Leiden, Netherlands (hereinafter referred to as Molecucular Probes) or Sigma-Aldrich-Fluka (Taufkirchen, Germany). examples for the synthesis of a nuc macromolecule with a low molecular weight Marker is given in Example 19, 20, 23, 36, 37, 38.

To the incorporation of the nucleotide portion in the nucleic acid is the linker coupled dye at a large distance from the nucleic acid strand, what to reduce the influences of the nucleobases on the fluorescence properties of the dye leads. Fluorescence yields individual Dyes are thus less of the local composition the nucleic acid sequence affected which leads to uniform signal intensity.

1.3.3.3.2.2 Structures high mass (macromolecules)

1.3.3.3.2.2.1 Nanocrystals

For example, Quantum dots. It can in a marker component Quantum dots with the same or different spectral properties US Pat. No. 6,322,901, US Pat. No. 6,423,551, US Pat. No. 6,251,303, US Pat. US Pat. 5,990,479).

1.3.3.3.2.2.2 Nano or Micro-particles

In which the largest dimensions of this Particles from 1nm to 2nm, from 2nm to 5nm, from 5nm to 10nm, from 10nm to 20nm, from 20nm to 50nm, from 50nm to 100nm, from 100nm up to 200nm, from 200nm to 500nm, from 500nm to 1000nm, from 1000nm to 5000 nm. The material of the particles can be pure, for example Metals such as gold, silver, aluminum (for example particles with surface plasmon resonance), - protein-Au conjugates: J. Anal. Chem. 1998, V. 70, p. 5177, - nucleic acid-Au conjugates: J. Am. Chem. Soc. 2001, V. 123, p.5164, J. Am. Chem. Soc. 2000, V. 122, p. 9071, Biochem. Biophys. Res. Commun 2000, V. 274, p. 817, Anal. Chem. 2001, V. 73, p. 4450), - latex (e.g., latex nanoparticles, Anal. Chem., 2000, V. 72, p., 1979) - plastic (Polystyrene), - paramagnetic Compounds / Mixtures Zhi ZL et al. Anal Biochem. 2003 Jul 15; 318 (2): 236-43, Dressman D et al. Proc Natl Acad Sci U S A. 2003 Jul 22; 100 (15): 8817-22 "- Metal Particles, - Magnetic Compounds / Mixtures Jain KK. Expert Rev Mol Diagn. 2003 Mar; 3 (2): 153-61, Patolsky F et al. Angew Chem Int Ed Engl. 2003 May 30; 42 (21): 2372-2376, Zhao X et al. Anal Chem. 2003 Jul 15; 75 (14): 3144-51, Xu H et al. J Biomed Mater Res. 2003 Sep 15; 66A (4): 870-9, JOSEPHONE Lee et al. US2003092029, KING-KAY-OLIVER et al. WO0119405.

1.3.3.3.2.2.3 Protein molecules

Out the following groups: enzymes (e.g., peroxidase, alkaline phosphatase, Urease, beta-galactosidase), - fluorescent Proteins (e.g., GFP), antigen-binding proteins (e.g., antibodies, tetramers, Affibodies (Nord et al., Al Nature Biotechnology, V. 15 (S.772-777)). or their components (e.g., Fab fragments), nucleic acid binding agents Proteins (e.g., transcription factor)

1.3.3.3.2.2.4 Nucleic acid chains

The nucleic acid chains including the group, oligonucleotides, modified oligonucleotides, wherein the nucleic acid chains a length from 10 to 20, from 20 to 50, from 50 to 100, from 100 to 200, from 200 to 500, from 500 to 1000, 1000 to 5,000, from 5,000 to 10,000, have from 10,000 to 100,000 nucleotides. It can use DNA, RNA, PNA molecules become. nucleic acid chains can zusäzliche Wear modifications such as free amino groups, dyes and other signaling molecules, e.g. macromolecular substances, such as enzymes or nanocrystals (MWG-Biotech, Detschland).

Further examples of macromolecules or macromolecule complexes that can be used in the present invention as units in the signal amplification marker component are disclosed in U.S. Pat US 4882269 . US 4687732 , WO 8903849, US 6017707 described.

1.3.3.3.3 The core component the marker component

The Core component has the function of multiple structural elements of Nuc-macromolecules bind. Preferably, several nucl linker components or more Marker units coupled to the core component.

1.3.3.3.3.1 Components

In an embodiment The core component consists of a water-soluble polymer, it can the polymer consist of the same or different monomers.

Examples of the core part components are derivatives of the following polymers:
Polyamides (eg polypeptides), polyacrylic acid, polyacrylamides, polyvinyl alcohols, nucleic acids, proteins. These polymers may be linear, globular, eg streptavidin or avidin, or branched, eg dendrimers. Crosslinked soluble polymers, such as, for example, crosslinked polyacrylamides (crosslinker bisacrylamide in combination with polyacrylamide), are used.

The Kern-Komponennte has several coupling points to the other elements can be bound e.g. structural marker units or nuc-linker component.

For example Poly-lysine molecules have multiple free amino groups to which multiple dye molecules, biotin molecules, hapten molecules or nucleic acid chains can be coupled. Poly-lysines have different molecular weights, e.g. 1000-2000, 2000-10000, 10000-50000 There you can for sale be acquired.

When another example of the core component serve nucleic acid strands, wherein the nucleic acid chains a length from 10 to 20, from 20 to 50, from 50 to 100, from 100 to 200, from 200 to 500, from 500 to 1000, 1000 to 5000, from 5000 to 10000 Have nucleotides. These nucleic acids serve as binding partners for sequence-complementary marker units.

In a further embodiment if the core component consists of a dendrimer, e.g. polypropylenimines, Polyaminoamines. examples for Other dendrimers are known: Cientifica "Dendrimers", 2003, Technology white papers No. 6, Klajnert et al. Acta Biochimica Polonica, 2001, v. 48, p. 199, Manduchi et al. Physiol. Genomics 2002, V.10, p.169, Sharma et al. Electrophoresis. 2003, V. 24, p. 2733, Morgan et al. Curr Opin Drug Discov Devel. 2002 Nov; 5 (6): 966-73, Benters et al. Nucleic Acids Res. 2002 Jan 15; 30 (2): E10, Nilsen et al. J Theor Biol. 1997 Jul 21, 187 (2): 273-84. Many dendrimers are commercially available (Genisphere, www.genisphere.com, Chimera Biotech GmbH).

Further Combinations for the core components of the components shown above are obvious to a person skilled in the art.

1.3.3.3.3.2 Coupling of the Marker units to the core component

structural Marker units can to the core component or to the linker component through a covalent bond, for example about a cross-linker (Chemistry of protein conjugation and cross-linking, S. Wang, 1993, ISBN 0-8493-5886-8, "Bioconjugation: protein coupling techniques for the biomedical sciences ", M. Aslam, 1996, ISBN 0-333-58375-2), or over an affine coupling, for example, biotin-streptavidin compound or hybridization of nucleic acid strands or Antigen-antibody interaction ( "Bioconjugation: protein coupling techniques for the biomedical sciences ", M. Aslam, 1996, ISBN 0-333-58375-2).

The Coupling of marker units to the core component takes place in an embodiment already during the synthesis of nuc macromolecules.

In another embodiment takes place first the chemical synthesis of nuc macromolecules, at where the marker component consists only of the core component. The coupling of marker units to the core component takes place only after the incorporation of nuc-macromolecules into the nucleic acid chain. By a big Number of potential binding sites at the core part is the probability the binding of the marker units to the core component and thus to the built-Nuk component much larger compared to conventional Nucleotide structures.

The Coupling chemistry depends in detail on the structure of the marker units and the structure of the core component from.

Covalent coupling:

The Link between the marker units and the core component can be found in an embodiment resistant (Example 33), e.g. at temperatures up to 100 ° C, for pH ranges between 3 and 12, and / or resistant to hydrolytic enzymes (e.g., esterases). In another embodiment of the invention the connection between the nuc-component and the linker below mild conditions fissile.

Examples for the Coupling of nucleic acids on dendrimers (corresponds to a coupling of marker units to a core component) are e.g. in Shchepinov et al. Nucleic Acids Res. 1999 Aug 1; 27 (15): 3035-41, Goh et al. Chem Commun (Camb). 2002 Dec 21; (24): 2954.

1.3.3.3.3.3 Coupling between Linker and marker

In an embodiment is the Nuk-Linker component directly to the signaling or signal-conveying Marker unit bound to the marker component.

In a further embodiment are one or more nucl linker components to the core component tied to the marker.

The link between the linker and the core component or the linker and the marker Component depends on the particular structure of the marker units or the structure of the core component. Binding may be both covalent and affine. "Bioconjugation: protein coupling techniques for biomedical sciences", M. Aslam, 1996, ISBN 0-333-58375-2.

Covalent coupling:

The Connection between the marker units and the core component can be in one embodiment resistant be, e.g. at temperatures up to 130 ° C, for pH ranges between 1 and 14, and / or resistant to hydrolytic enzymes (e.g., proteases, Esterases). In another embodiment of the invention the connection between the nuc-component and the linker below mild conditions fissile.

1.3.3.3.4 The ratio of Nuc components in a nuc macromolecule

In a nuc macromolecule can 1-1000 Nuk components, s.o. The distribution can be Nuk components in a nuc-macromolecule population are, and do not have to vary necessarily represent a constant value for each nuc-macromolecule.

In an embodiment all have nuc macromolecules an equal number of nuc components per a nuc macromolecule. For example, you can maximum for one strepavidin molecule 4 biotin molecules be bound at saturating Concentration of nucl linker components, a uniform population on nuc macromolecules arises.

In another embodiment have the nuc macromolecules population has a defined average number of nuclei Components per nuc-macromolecule, in the Population itself, however, finds a distribution of actual Occupation of the nuc macromolecules with Nuk components instead. The distribution information of nuc components per nuc-macromolecule in this case represent an average. Such nuc-macromolecules may be, for example be used when the nuc-component has a terminating Has property.

1.3.3.3.5 The ratio of Marker units in a nuc macromolecule

In a nuc macromolecule can 1-1000 marker units occur, s.o. The distribution can be of marker units in a nuc-macromolecule population do not and do not fluctuate necessarily represent a constant value for each nuc-macromolecule.

In an embodiment all have nuc macromolecules an equal number of marker units per one nuc macromolecule. For example can per one strepavidin molecule a maximum of 4 biotin molecules be bound, avidin biotin technology, Methods in Enzymology v.184, 1990.

In another embodiment have the nuc macromolecules a population a defined average number of marker units per nuc-macromolecule, in the population itself, however, finds a distribution of the actual Occupation of the nuc macromolecules held with marker units. At saturating concentrations The synthesis of marker components is becoming increasingly uniform Occupation of the nuc macromolecules held with marker units.

Such Nuc-macromolecules are for example Methods of importance in which fluctuations in the signal intensity within one Nuc-macromolecule population are negligible.

So plays for example in areas, where it concerns the qualitative proof is the exact number of marker units per nuc-macromolecule a subordinate Role. In such cases the presence of a stable signal is important. examples for such methods are single molecule sequencing methods.

It It should be apparent to one skilled in the art that the recited marker components are a significant larger molecule size and mass have, as the respective nuk component itself. Furthermore, should other examples of macromolecular marker components to a person skilled in the obvious.

1.3.3.3.6 Substrate properties the nuc macromolecules

The Nuk component can serve as a substrate for different enzymes. For example, the nuc-component is used in this embodiment as nucleoside triphosphate, as a substrate for a polymerase so that the Nuk component in a growing strand by a polymerase can be incorporated and thus the entire nuc-macromolecule to the Strand is covalently coupled.

Further examples for Enzymes are kinases, phosphorylases, transferases.

The Determine substrate properties of the nuc-component (s) the substrate properties of the nuc macromolecules. So can the nuk component serve as a terminator, so that only a single nuc-macromolecule incorporated can be. In another embodiment, the nuc-component is used as a reversible terminator, which controlled the implementation of a gradual extension reaction allowed, such as in Tcherkassov WO 02088382 is shown.

1.3.3.3.7 Function of the marker

The Macromolecular marker component may be in one embodiment a signaling, signal-transmitting, catalytic or affine Have function.

at contains the signaling function the marker component constituents already during the chemical synthesis to nuc-macromolecules are bound, s. example 33rd

at the signal-transmitting function carries the marker component components, the first through a reaction with signaling molecules their Develop signal properties, s. Example 32.

For example can several biotin molecules, e.g. 100, make up the marker part. After incorporation of the nuc-macromolecules takes place a detection reaction with modified streptavidin molecules. In In another example, the nucleic acid chains have the signal-transmitting Function. After incorporation of nuc-macromolecules, hybridization occurs of uniform oligonucleotides with detectable units, e.g. Fluorescent dyes (MWG Biotech) to the marker component. In one further examples have amino or mercapto groups, for example 50 amino groups per marker, the signal-transmitting function. After this Incorporation of nuc macromolecules into the nucleic acid chain a chemical modification with reactive components, e.g. with dyes, such as for incorporated allyl-amino-dUTP described, Diehl et al. Nucleic Acid Research, 2002, V. 30, No. 16 e79.

In another embodiment the macromolecular marker component has a catalytic function (in the form of an enzyme or ribozyme). It can be different enzymes can be used, e.g. Peroxidase or alkaline phosphatase. thanks the coupling to the nuc-component, the respective enzyme by the incorporation of nuc macromolecules to the nucleic acid strand covalently bound.

In a further embodiment the macromolecular marker component has an affinity functionality to one another molecule. examples for such markers form streptavidin molecules or nucleic acid chains, Example 30 or 32.

1.3.4. low molecular weight marker

To the State of the art Labeling of nucleotides, for example, with one or two biotin molecules or two dye molecules, one or two hapten molecules (e.g., digoxigenin).

1.3.5. Conventionally modified nucleotide

One Nucleotide with a linker (average length between 5 and 30 atoms) and a marker. Usually enters conventionally modified nucleotide a low molecular weight marker, e.g. a dye or a biotin molecule.

1.3.6 Anchor function

Refers to the ability of nucleic acid chains fixed on the solid phase to bind other complementary nucleic acid chains. The process is called hybridization. This bond becomes too affi counted bindings.

1.3.7 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.

1.3.8 Production of 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.

1.3.9 Incubation step

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".

1.3.10 DNA

• deoxyribonucleic acid of different origin and length (oligonucleotides, PCR products, Plasmids, genomic DNA, cDNA, ssDNA, dsDNA).
• RNA - ribonucleic acid

1.3.11 dNTP

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

1.3.12 Marked components

Under labeled components are understood to be molecules that are detectable Wear marker. Preferred are marked in this application Nucleotides (conventionally modified nuclides as well as nuc macromolecules) and labeled nucleic acid chains considered. The labeled components may also have other modifications include.

1.3.13 NTP

• Nucleoside triphosphates, substrates for RNA polymerases

1.3.14 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 becomes abbreviations the majority formed by using the suffix "s", "NT" is for example for "nucleotide", "NTs" stands for several Nucleotides.

1.3.15 NT *

• Modified nucleotide (conventionally modified nucleotide or a nuc macromolecule), NTs * means: modified nucleotides

1.3.16 NSK

• Nucleic acid chain.

1.3.17 NSKF

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

1.3.18 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.

1.3.19 Polymerases

• Enzymes that are complementary Nucleotides into a growing DNA or RNA strand (e.g., DNA polymerases, Reverse transcriptase, RNA polymerases)

1.3.20 primer binding site (PBS)

• Part of the sequence in the NSK or NSKF to which the primer binds.

1.3.21 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.

1.3.22 Regenerated surface

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

1.3.23 Tm

• melting temperature

1.3.24 TCEP

• Tris-carboxy-ethyl-phosphine

1.3.25 non-specific Binding of marked components

Degree of nonspecific binding is given as the number of binding events (mean) per 100 μm ² . 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.

1.3.26 Wide Field Optics

Under Weitfeldoptik detection devices are projected, the resolution in the range of have over 0.2 microns, for example, a resolution of 0.5 microns or 1 micron. This generic term distinguishes itself from near-field optics, which enable a 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 5.1008, 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.

1.4 Object of the invention:

The The object of the present invention is therefore a method for sequence analysis of nucleic acid chains to provide that does not have the disadvantages of the above-mentioned methods and above all a cheaper, faster and more efficient analysis of nucleic acid sequences allows. In particular, the method should be capable of many sequences to be determined in parallel.

The The object of the invention was also a surface of a provide solid phase in the analysis of nucleic acid chains can be used and a low non-specific binding of both labeled and unlabeled nucleotides as well as labeled and unlabeled 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.

1.5 Short 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 enzymatischer Einbau von markierten Nukleotiden an den 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

wobei man die relative Position einzelner Matrize-Primer-Komplexe auf der Reaktionsoberfläche und die Sequenz dieser Matrizen durch spezifische Zuordnung der in Stufe 3.3 in aufeinanderfolgenden Zyklen an den jeweiligen Positionen detektierten Fluoreszenzsignale zu den NTs bestimmt.The object is achieved by a method for highly parallel sequence analysis of nucleic acid sequences with optical means (nucleic acid chains, NSKs), which 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 enzymatic incorporation of labeled nucleotides on 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

whereby the relative position of individual template-primer complexes on the reaction surface and the sequence of these templates are determined by specific assignment of the fluorescence signals to the NTs detected in step 3.3 in successive cycles at the respective positions.

marked Nucleotides can both conventionally labeled or modified nucleotides as well as nuc macromolecules represent.

Out The determined partial sequences can be, for example, the overall sequence the nucleic acid chain determine. Under a parallel sequence analysis is in this Context understood the simultaneous sequence analysis of many NSKFs (for example, 1,000,000 to 10,000,000), these NSKFs being from a uniform NSK population or of several different 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 application describes a surface of a solid phase for the method according to the invention which has a very low unspecific binding of labeled components such as for example labeled nucleotides and labeled nucleic acids ( 6A -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 solid phase surface of the invention with fixed nucleic acids can be used in methods for the analysis of the nucleic acids, the hybridization of complementary labeled nucleic acids and / or enzymatic reactions on fixed nucleic acid chains ( 1 , 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 are randomly distributed on the surface during immobilization or binding, ie So you do not have to pay attention to an exact positioning of the individual chains. The NSKF primer complexes must be fixed on the surface in such a density that an unambiguous assignment of the later detected signals from the incorporated labeled nucleotides to individual NSKFs is ensured.

• 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.

• – Verfahren zur Sequenzierung
• – 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
• – Nuk-Makromoleküle, die im Sequenzierverfahren eingesetzt werden können.

The following topics are displayed in the application:

• - Method for sequencing
• 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
• - Nuc-macromolecules that can be used in the sequencing process.

• 1. Ein Verfahren zur parallelen Sequenzanalyse von Nukleinsäuresequenzen (Nukleinsäureketten, NSKs) mit optischen Mitteln, das folgende Schritte einschließt: 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 enzymatischer Einbau von markierten Nukleotiden an den 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
• 2. Feste Phase für Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekt 1, 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) oder mit photolabilen Schutzgruppen (US Pat. Nr. 5753788, US Pat. Nr. 5831070).
• 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 10 μm 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. 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 nach Aspekt 1, wobei die feste Phase nach Aspekten 2 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 1,3 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 1,5 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 105, wobei als markierte Nukleotide konventionell modifizierte Nukleotide eingesetzt werden.
• 107. Verfahren zur parallelen Analyse einer Vielzahl einzelner Nukleinsäuremoleküle mit optischen Mitteln nach Aspekt 60 bis 105, wobei als markierte Nukleotide Nuk-Makromoleküle eingesetzt werden.
• 108. 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.
• 109. Verfahren zur Analyse von Nukleinsäureketten mit der festen Phase nach Aspekten 2 bis 59, wobei die zu analysierenden Nukleinsäureketten an der festen Phase fixiert sind und die Analyse durch die Hybridisierung von komplementären markierten Nukleinsäureketten erfolgt.
• 110. Verfahren zur Analyse von Nukleinsäureketten mit der festen Phase nach Aspekten 2 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.

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

• 1. A method for parallel sequence analysis of nucleic acid sequences (nucleic acid chains, NSKs) with optical means, comprising 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 Performing a cyclic reaction with the solid phase-fixed primer-template complexes, including the following steps: 3.1 enzymatic incorporation of labeled nucleotides on 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 optionally 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
• 2. Solid phase for methods for parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspect 1, 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 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 the solid phase. 2. coupling nucleic acid chains to the regenerated surface of the solid phase
• 18. A method for producing the solid phase according to Aspects 1 to 16, wherein the preparation is as follows Steps include: 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 nucleic acid chains to the linker component
• A method of preparing the solid phase of aspects 1 to 16, wherein the preparation includes 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
• 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) or with photolabile protecting groups (US Pat. No. 5,753,788, US Pat. No. 5,831,070).
• 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. Method for producing the solid phase according to aspect 26, wherein the chemical reaction in estate unit 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 outer layer is 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 extender-capable primer-template complexes is preferably in one of the following ranges: 1/100 μm ² to 10/100 μm ² , 10/100 μm ² to 20/100 μm ² , 20/100 μm ² to 30/100 μm 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 . 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 according to aspect 1, wherein the solid phase according to aspects 2 to 59 is used.
• 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. A method for the parallel analysis of a plurality of individual nucleic acid molecules with 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 up to 1.3 microns
• 80. A method for 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
• 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. Method for parallel analysis of a plurality of individual nucleic acid molecules by optical means according to aspects 60 to 82, wherein the nucleic acid molecules to be analyzed represent nucleic acid chains having 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. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to aspects 60 to 84, wherein the nucleic acid molecules to be analyzed represent 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 for 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 105, wherein as a labeled nucleotides conventionally modified nucleotides are used.
• 107. Method for the parallel analysis of a multiplicity of individual nucleic acid molecules with optical means according to aspects 60 to 105, wherein nuc-macromolecules are used as labeled nucleotides.
• 108. 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.
• 109. A method for the analysis of nucleic acid chains with the solid phase according to aspects 2 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.
• 110. A method of analyzing nucleic acid chains with the solid phase of aspects 2 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.

in the Related to aspects 1 to 110 of the invention are nuc-macromolecules and their use in the sequencing method according to aspects 1, 60 to 105 as additional aspects 1 to 64 of the invention.

Nuk

– ein Nukleotid oder ein Nukleosid ist (Nuk-Komponente)

Linker

– eine Linker-Komponente, die folgende Bestandteile einschließt: a) Kopplungseinheit L – ein Teil des Linkers, das die Verbindung zwischen Nuk und dem Linkerrest darstellt b) Polymer – ein Teil des Linkers, das ein wasserlösliches Polymer mit einer durchschnittlichen Länge zwischen 100 und 10.000 Atome ist c) Kopplungseinheit T – ein Teil des Linkers, das die Verbindung zwischen dem Linkerrest und dem Marker darstellt

Marker

– eine Marker-Komponente ist

n

– eine ganze Zahl zwischen 1 und 100 ist

1. Additional aspect of the invention relates to macromolecular compounds having the structure:
(Nuk linker) _n marker
in which:

Nuk

Is a nucleotide or a nucleoside (nuc-component)

left

A linker component which includes the following components: a) coupling unit L - a part of the linker which represents the link between Nuk and the linker radical b) polymer - a part of the linker comprising a water-soluble polymer with an average length between 100 and 100 10,000 atoms is c) coupling unit T - a part of the linker that represents the link between the linker residue and the marker

marker

- is a marker component

n

- is an integer between 1 and 100

A further additional aspect 2 of the invention relates to macromolecular compounds according to additional aspect 1, wherein the nuc-component includes the following structures ( 17A ):
In which:
Base - is independently selected from the group of adenine, or 7-deazaadenine, or guanine, or 7-deazaguanine, or thymine, or cytosine, or uracil, or their modifications, where L is the link between the nuc-component and the linker Component represents (coupling unit L) and X is the coupling point of the coupling unit L at the base
R ₁ - is H
R ₂ - is independently selected from the group H, OH, halogen, NH ₂ , SH or protected OH group
R ₃ - is independently selected from the group H, OH, Hal, PO ₃ , SH, NH ₂ , O-R _3-1 , P (O) _{m -R 3-1} , NH-R _3-1 , SR _{3 -1} , Si-R _3-1 wherein R _{3-1 is} a chemically, photochemically or enzymatically cleavable group and m is 1 or 2.
R ₄ - is H or OH
R ₅ - is independently selected from the group OH, or a protected OH group, or monophosphate group, or diphosphate group or triphosphate group or polyphosphates

A further aspect 3 of the invention relates to macromolecular compounds according to additional aspect 1, wherein the nuc-component includes the following structures ( 17B ):
In which:
Base - is independently selected from the group of adenine, or 7-deazaadenine, or guanine, or 7-deazaguanine, or thymine, or cytosine, or uracil, or their modifications capable of enzymatic reactions
R ₁ - is H
R ₂ - is independently selected from the group H, OH, Hal, NH ₂ , SH or protected OH group
R ₃ - is independently selected from the group O-R _3-2 , P (O) _m - R _3-2 , where m is 1 or 2, NH-R _3-2 , SR _3-2 , Si-R _{3 -2} or, wherein R _{3-2 is} the coupling site of the coupling unit L to the nucleotide
Coupling unit L - the connection between the nuc-component and the linker component
R ₄ - is H or OH
R ₅ - is independently selected from the group OH, or a protected OH group, or monophosphate group, or diphosphate group or triphosphate group

A further additional aspect 4 of the invention relates to macromolecular compounds according to additional aspect 1, wherein the nuc-component includes the following structures ( 17B ):
In which:
Base - is independently selected from the group of adenine, or 7-deazaadenine, or guanine, or 7-deazaguanine, or thymine, or cytosine, or uracil, or their modifications capable of enzymatic reactions
R ₁ - is H
R ₂ - is independently selected from the group H, OH, Hal, NH ₂ , SH or protected OH group
R ₃ - is independently selected from the group H, OH, Hal, PO ₃ , SH, NH ₂ , O-R _3-1 , P (O) m -R _3-1 , NH-R _3-1 , SR _{3 -1} , Si-R _3-1 wherein R _{3-1 is} a chemically, photochemically or enzymatically cleavable group and m is 1 or 2.
R ₄ - is H or OH
R ₅ - is independently selected from the group O-R _5-1 -L, or P- (O) ₃ - R _5-1 -L (modified monophosphate group), or P- (O) ₃ -P- ( O) ₃ -R _5-1 -L (modified diphosphate group)
or P- (O) ₃ -P- (O) _3- P- (O) _{3 -R 5-1-} L (modified triphosphate group), wherein R _{5-1 is} the coupling site of the coupling moiety L to the nucleotide, and Coupling unit L is the connection between the nuc-component and the linker component.

A further aspect 5 of the invention relates to macromolecular compounds according to additional aspects 1 to 4, wherein coupling unit L includes the following structural elements:
R ₆ -NH-R ₇ , R ₆ -OR ₇ , R ₆ -SR ₇ , R ₆ -SS-R ₇ , R ₆ -CO-NH-R ₇ , R ₆ -NH-CO-R ₇ , R ₆ -CO-OR ₇ , R ₆ -O-CO-R ₇ , R ₆ -CO-SR ₇ , R ₆ -S-CO-R ₇ , R ₆ -P (O) ₂ -R ₇ , R ₆ -Si R ₇ , R ₆ - (CH ₂ ) _n -R ₇ , R ₆ - (CH ₂ ) _n -R ₇ , R ₆ -A- (CH ₂ ) _n -R ₇ , R ₆ - (CH ₂ ) _n -BR ₇ ,
R ₆ - (CN = CH-) _n -R ₇ , R ₆ - (A-CH = CH-) _n -R ₇ , R ₆ - (CH = CH-B-) _n -R ₇ , R ₆ - ( CH = CH-CH ₂ -B-) _n -R ₇ , R ₆ -A-CH = CH- (CH ₂ -) _n -R ₇ , R ₆ - (- CH = CN-CH ₂ ) -BR ₇ ,
R ₆ - (C≡C-) _n -R ₇ , R ₆ - (AC≡C-) _n -R ₇ , R ₆ - (AC≡C-CH ₂ ) -R ₇ , R ₆ - (C≡CB -) _n -R ₇ ,
R ₆ - (C≡C-CH ₂ -B-) _n -R ₇ , R ₆ -AC≡C- (CH ₂ -) _n -R ₇ , R ₆ - (- C≡C-CH ₂ ) _n - BR ₇ ,
R ₆ - (- C≡C-CH ₂ -CH ₂ ) -BR ₇
wherein R ₆ - is the nuc-component, R ₇ - is the linker radical and A and B independently include the following structural elements: -NH-, -O-, -S-, -SS-, -CO-NH-, -NH -CO-, -CO-O-, -O-CO-, -CO-S-, -S-CO-, -P (O) ₂ -, -Si-, - (CH ₂ ) _n -, a photolabile Group, where n - is 1 to 5

One additional aspect 6 of the invention relates to macromolecular compounds according to additional aspects 1 to 5, wherein the linker component is a water-soluble Polymer includes.

A further additional aspect 7 of the invention relates to macromolecular compounds according to additional aspect 6, wherein the linker component includes water-soluble polymers independently selected from the following group:
Polyethylene glycol (PEG), polysaccharides, dextran, polyamides, polypeptides, polyphosphates, polyacetates, poly (alkylene glycols), copolymers of ethylene glycol and propylene glycol, poly (olefinic alcohols), poly (vinyl pyrrolidones), poly (hydroxyalkyl methacrylamides), poly (hydroxyalkyl methacrylates) , Poly (x-hydroxy acids), polyacrylic acid, polyacrylamide, poly (vinyl alcohol).

One Further additional aspect 8 of the invention relates to macromolecular compounds according to additional aspects 1 to 7, wherein the linker component is an average Length between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 10,000, 10,000 to 100,000 atoms.

One Further additional aspect 9 of the invention relates to macromolecular compounds according to additional aspects 1 to 8, wherein a marker component is a signaling, has signal-transmitting, catalytic or affine function

One Further additional aspect 10 of the invention relates to macromolecular Compounds according to additional aspects 1 to 9, wherein a marker component consists of a structural marker unit

One Further additional aspect 11 of the invention relates to macromolecular Compounds according to additional aspects 1 to 9, wherein a marker component consists of several structural marker units bound to a core component

A further aspect 12 of the invention relates to macromolecular compounds according to additional aspects 10 or 11, wherein a structural marker unit independently includes one of the following structural elements:
Biotin, hapten, radioactive isotope, rare earth element, dye, fluorescent dye.

A further aspect 13 of the invention relates to macromolecular compounds according to additional aspects 10 or 11, wherein a structural marker unit independently includes one of the following elements:
Nanocrystals or their modifications, proteins or their modifications, nucleic acid chains or their modifications, particles or their modifications.

A further additional aspect 14 of the invention relates to macromolecular compounds according to additional aspect 13, wherein a structural marker unit includes one of the following proteins:
Enzymes or their conjugates or modifications,
Antibodies or their conjugates or modifications,
Streptavidin or its conjugates or modifications,
Avidin or its conjugates or modifications

One additional aspect 15 of the invention relates to macromolecular Compounds according to additional aspect 13, wherein a structural marker unit one of the following types of nucleic acid chains: DNA, RNA, PNA, where the length of nucleic acid chains between 10 and 10,000 nucleotides or their equivalents.

One additional aspect 16 of the invention relates to macromolecular Compounds according to additional aspects 11 to 15, wherein the core component the marker component independently includes one of the following elements: water-soluble polymer independently selected from the group of: polyamides (e.g., polypeptides), polyacrylic acid and their derivatives, polyacrylamides and their derivatives, polyvinyl alcohols and their derivatives, nucleic acid chains and their derivatives, streptavidin or avidin and their derivatives, Dendrimers, wherein individual polymers linear or branched or under can be networked

One additional aspect 17 of the invention relates to macromolecular Compounds according to additional aspects 1 to 9, 11 to 16, wherein the compound between several structural marker units and the core component covalent or affine occurs.

One Further additional aspect 18 of the invention relates to macromolecular Compounds according to additional aspects 1 to 10, wherein the compound between a structural marker unit and the linker covalent or Affine happens.

One Further additional aspect 19 of the invention relates to macromolecular Compounds according to additional aspects 1 to 9, 11 to 17, wherein the compound between the core component and the linker covalent or affine he follows

One additional aspect 20 of the invention relates to macromolecular Compounds according to additional aspects 1 to 19, wherein only one nuc-component with a coupled linker component to the marker component is bound, where the linker length between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 5000 atoms.

Another additional aspect 21 of the invention relates to macromolecular compounds according to additional aspects 1 to 20, wherein only one Nuk component with a coupled linker component to the Mar ker component, wherein the linker length is between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 5000 atoms and the linker component includes one or more compounds cleavable under mild conditions.

One additional aspect 22 of the invention relates to macromolecular Compounds according to additional aspects 1 to 21, wherein only one nuc-component with a coupled linker component to the marker component is bound, where the linker length between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 5000 atoms and one or more parts of the nuc macromolecule so are modified that only a nuk component can be incorporated into a growing strand can.

One additional aspect 23 of the invention relates to macromolecular Compounds according to additional aspects 1 to 19, wherein several nuc-components each over a linker are coupled to a marker component, wherein the Length of respective linker component between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 5000 atoms.

One Further additional aspect 24 of the invention relates to macromolecular Compounds according to additional aspects 1 to 19, 23, wherein several nuc-components each have a Linker coupled to a marker components, with the length of the respective linker component between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 5000 atoms and the respective linker one or more cleavable under mild conditions Includes connections.

One additional aspect 25 of the invention relates to macromolecular Compounds according to additional aspects 1 to 19, 23, 24, wherein several Nuc-components each over a linker coupled to a marker component, the length of the respective linker component between 50 to 100, 100 to 200, 200 to 500, 500 to 1000, 1000 to 2000, 2000 to 5000 atoms and a or several parts of the nuc macromolecule are so modified that only one Nuk component can be incorporated into a growing nucleic acid chain.

One Further additional aspect 26 of the invention relates to oligonucleotides or polynucleotides which include at least one nuc-macromolecule by additional aspect 1 to 25 per one nucleic acid chain lock in.

One additional aspect 27 of the invention relates to oligonucleotides or polynucleotides according to additional aspect 26, wherein oligo- or polynucleotides RNA, DNA or PNA are their length between 5 and 50,000 nucleotides.

One additional aspect 28 of the invention relates to a method for Modification of nucleic acid chains, being for the coupling nuc-macromolecules after Additional aspects 1 to 25 are used

• – mindestens eine Art der Nuk-Makromoleküle oder deren Zwischenstufen nach Zusatzaspekten 1 bis 25, wobei jede Art der Nuk-Makromoleküle eine für sie charakteristische Markierung besitzt
• – mindestens eine Population der Nukleinsäureketten,
• – mindestens eine Enzymart zur Kopplung von Nuk-Makromoleküle an die Nukleinsäureketten,

A further aspect 29 of the invention relates to a process according to additional aspect 28, wherein the modification is effected by an enzymatic coupling and the reaction mixture includes the following components:

• At least one kind of the nuc-macromolecules or their intermediates according to additional aspects 1 to 25, wherein each type of nuc-macromolecule has a characteristic marking for them
• At least one population of the nucleic acid chains,
• At least one type of enzyme for coupling nuc macromolecules to the nucleic acid chains,

• – mindestans eine Art der Nuk-Makromoleküle oder deren Zwischenstufen nach Zusatzaspekten 1–25, wobei jede Art der Nuk-Makromoleküle eine für sie charakteristische Markierung besitzt
• – mindestens eine Population der Nukleinsäureketten,
• – mindestens eine Enzymart zur Kopplung von Nuk-Makromoelküle an die Nukleeinsäureketten
• – mindestens eine weitere Art von Nukleosidtriphosphaten

A further aspect 30 of the invention relates to a process according to additional aspect 28, wherein the modification is effected by an enzymatic coupling and the reaction mixture includes the following components:

• At least one kind of the nuc-macromolecules or their intermediates according to additional aspects 1-25, wherein each type of nuc-macromolecule possesses a characteristic marking for them
• At least one population of the nucleic acid chains,
• - At least one type of enzyme for the coupling of nuc-macromolecules to the nucleic acid chains
• - At least one other type of nucleoside triphosphates

One additional aspect 31 of the invention relates to a method according to Additional Aspects 29, 30, the type of enzyme independently being one of the following groups includes: DNA polymerase, RNA polymerase, terminal transferase,

One Further additional aspect 32 of the invention relates to a method according to Additional aspect 30, the "other Type of nucleoside triphosphates independently selected is from the group of: ribo-nucleoside triphosphates (ATP, GTP, UTP, CTP), from 2'-deoxyribonucleoside triphosphates (dATP, dUTP, dTTP, dCTP, dGTP) of 2 ', 3'-dideoxynucleoside triphosphates (ddATP, ddGTP, ddUTP, ddCTP, ddTTP).

One Further additional aspect 33 of the invention relates to a method according to Additional aspect 32, the "other Type of nucleoside triphosphates are conventionally modified nucleotides with a label, this label being independent selected is from the group of: a fluorescent dye, a biotin, a hapten, a radioactive element.

One Further additional aspect 34 of the invention relates to a method according to Additional aspects 28 to 33, where at least two different Populations of nucleic acid chains present are

One Further additional aspect 35 of the invention relates to a method according to Zusatzaspekt 34, wherein at least one of the populations of nucleic acid chains has a primer function and at least one pupulation of the nucleic acid chains a matrix function.

One additional aspect 36 of the invention relates to a method according to Zusatzaspekt 28, wherein the modification by a chemical coupling takes place, wherein the coupling of nuc-macromolecules to nucleic acid chains by phosphoroamidite coupling takes place.

One additional aspect 37 of the invention relates to a method according to Additional aspects 28 to 36, wherein Nuk macromolecules used in the labeling process Be the coupling of only a single Nuk component in the growing nucleic acid strand allow multiple installation by modifications to the Nuk component, and / or the linker component and / or the marker component prevented becomes.

One additional aspect 38 of the invention relates to a method according to Zusatzaspekt 37, wherein the further coupling reversibly prevented becomes.

One Further additional aspect 39 of the invention relates to a method according to Zusatzaspekt 37, wherein the further coupling irreversibly prevented becomes.

One additional aspect 40 of the invention relates to a method according to Additional aspects 28 to 36, wherein Nuk macromolecules used in the labeling process Becoming a coupling of several Nuk component in the growing nucleic acid strand allow

One additional aspect 41 of the invention relates to a method according to Additional aspects 28-40, where the nucleic acid chains participating in the reaction coupled to a fixed phase and addressable positions to have.

One Further additional aspect 42 of the invention relates to a method according to Zusatzaspekt 41, wherein the nucleic acid chains a uniform Represent population

One Further additional aspect 43 of the invention relates to a method according to Zusatzaspekt 41, wherein the nucleic acid chains two or more represent different populations and each of the populations has an addressable position on the solid phase

One additional aspect 44 of the invention relates to a method according to Zusatzaspekten 41, 42, wherein the coupling of nuc macromolecules on a Population of uniform nucleic acid molecules fixed to the solid phase, wherein the marker component of the nuc macromolecule is elongated after coupling nucleic acid strand remains and is not separated.

One additional aspect 45 of the invention relates to a method according to Zusatzaspekten 41, 42, wherein the coupling of nuc macromolecules on a Population of uniform nucleic acid molecules fixed to the solid phase, wherein the marker component or its individual components with or without linker component of the nuc macromolecule during coupling or after the coupling of the incorporated into the growing nucleic acid strand nuc-component is separated.

Another additional aspect 46 of the invention relates to a method according to additional aspects 41, 43, wherein the coupling of nuc-macromolecules in a reaction mixture is carried out in parallel on two or more different populations of the solid phase of fixed nucleic acid molecules, these populations each having different addressable positions on the solid phase and extending the marker component of the nuc-macromolecule after the coupling on Nucleic acid strand remains and is not separated.

One additional aspect 47 of the invention relates to a method according to Zusatzaspekten 41, 43, wherein the coupling of nuc macromolecules in one Reaction batch parallel to two or more different Populations on the solid phase of fixed nucleic acid molecules take place, these populations have different addressable positions at the solid phase and the marker component or its individual Components with or without linker component of the nuc macromolecule during the Coupling or separated after the coupling of the nuc-component becomes.

One additional aspect 48 of the invention relates to a method according to Additional aspects 41 to 47, wherein the addressable positions with Nucleic acid molecules on the solid phase distributed as spots on a flat surface are, where per one spot nucleic acid molecules are uniform.

One Further additional aspect 49 of the invention relates to a method according to Additional aspects 41 to 47, wherein the addressable positions with Nucleic acid molecules to the globule or particles are attached, wherein per a bead nucleic acid molecules uniformly are.

One additional aspect 50 of the invention relates to a method according to Additional aspects 41 to 47, wherein the addressable positions with Nucleic acid molecules in one Multi vascular system, like microtiter plate or nanotiter plate or picotiter plate, are distributed, wherein in a vessel of the multi-vascular system the nucleic acid molecules uniform are.

• a) Bereitstellung mindestens einer Population an einzelsträngigen Nukleinsäureketten
• b) Hybridisierung von sequenzspezifischen Primern an diese Nukleinsäureketten, wobei extensionsfähige NSK-Primer-Komplexe entstehen
• c) Inkubation von mindestens einer Art der Nuk-Makromoleküle nach Zusatzaspekten 1 bis 25 zusammen mit einer Art der Polymerase nach Zusatzaspekt 31 mit den in Schritten a und b bereitgestellten NSK-Primer-Komplexen unter Bedingungen, die den Einbau von komplementären Nuk-Makromolekülen zulassen, wobei jede Art der Nuk-Makromoleküle eine für sie charakteristische Markierung besitzt.
• d) Entfernung der nicht eingebauten Nuk-Makromoleküle von den NSK-Primer-Komplexen
• e) Detektion der Signale von in die NSK-Primer-Komplexe eingebauten Nuk-Makromolekülen
• f) Entfernung der Linker-Komponente und der Marker-Komponente von den in die NSK-Primer-Komplexe eingebauten Nuk-Makromolekülen
• g) Waschen der NSK-Primer-Komplexe

gegebenenfalls Wiederholung der Schritte (c) bis (g),A further aspect 51 of the invention relates to a method according to additional aspects 28 to 35 and 37 to 50, which includes the following steps:

• a) Provision of at least one population of single-stranded nucleic acid chains
• b) hybridization of sequence-specific primers to these nucleic acid chains, wherein NSK primer complexes capable of extension are produced
• c) Incubation of at least one type of nuc-macromolecules according to additional aspects 1 to 25 together with a type of polymerase according to additional aspect 31 with the NSK-primer complexes provided in steps a and b under conditions which allow the incorporation of complementary nuc-macromolecules , wherein each type of nuc-macromolecule has a characteristic of their mark.
• d) Removal of unincorporated nuc-macromolecules from the NSK primer complexes
• e) detection of the signals of nuc-macromolecules incorporated into the NSK primer complexes
• f) Removal of the linker moiety and the label component from the nuc-macromolecules incorporated into the NSK primer complexes
• g) washing the NSK primer complexes

optionally repeating steps (c) to (g),

One additional aspect 52 of the invention relates to a method according to Additional aspects 28-40, wherein the nucleic acid chains to a solid phase in random Arrangement are coupled.

• a) zu den auf der Oberfläche gebundenen NSKF-Primer-Komplexen eine Lösung zugibt, die eine oder mehrere Polymerasen und ein bis vier Nuk-Makromoleküle enthält, die eine mit Fluoreszenzfarbstoffen markierte Marker-Komponente haben, wobei die bei gleichzeitiger Verwendung von mindestens zwei Nuk-Makromoleküle jeweils an die Marker-Komponente befindlichen Fluoreszenzfarbstoffe so gewählt sind, dass sich die verwendeten Nuk-Makromoleküle durch Messung unterschiedlicher Fluoreszenzsignale voneinander unterscheiden lassen, wobei die Nuk-Makromoleküle strukturell so modifiziert sind, dass die Polymerase nach Einbau eines solchen Nuk-Makromoleküls in einen wachsenden komplementären Strang nicht in der Lage ist, ein weiteres Nuk-Nakromolekül in denselben Strang einzubauen, wobei die Linker-Komponente mit der Marker-Komponente abspaltbar sind, 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 Nuk-Makromolekül verlängert werden, man
• c) die in Stufe b) erhaltene stationäre Phase unter Bedingungen wäscht, die zur Entfernung nicht in einen komplementären Strang eingebauter Nuk-Makromoleküle geeignet sind, man
• d) die einzelnen, in komplementäre Stränge eingebauten Nuk-Makromoleküle 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 Linker-Komponente und die Marker-Komponente von den am komplementären Strang angefügten Nuk-Komponenten abspaltet, man
• f) die in Stufe e) erhaltene stationäre Phase unter Bedingungen wäscht, die zur Entfernung der Marker-Komponente 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 Nuk-Makromolekülen bestimmt.Another additional aspect 53 of the invention relates to a method according to additional aspects 28 to 41, 52 for the parallel sequence analysis of nucleic acid sequences (nucleic acid chains, NSKs) in which
Generates fragments (NSKFs) of single-stranded NSKs of about 50 to 1000 nucleotides in length, which can be overlapping partial sequences of an overall sequence;
the NSKFs bind in a random array using a single or multiple different primers in the form of NSKF primer complexes on a reaction surface, the density of the surface-primed NSKF primer complexes providing optical detection of the signals from individual integrated nuclides. Macromolecules allows, one
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 nuc-macromolecules having a fluorescent dye-labeled marker component, wherein the simultaneous use of at least two nucl Macromolecules are each located on the marker component fluorescent dyes are selected so that the nuc-macromolecules used can be distinguished from one another by measuring different fluorescence signals, wherein the nuc-macromolecules are structurally modified such that the polymerase, after incorporation of such a nuc-macromolecule in a growing complementary strand, is unable to form another nuclacromolecule incorporate the same strand, wherein the linker component with the marker component are cleavable, one
• b) incubating the stationary phase obtained in step a) under conditions which are suitable for extending the complementary strands, wherein the complementary strands are each extended by one nuc-macromolecule;
• c) washing the stationary phase obtained in step b) under conditions which are suitable for removing nuc-macromolecules not incorporated into a complementary strand;
• d) the individual nucleated macromolecules incorporated into complementary strands are detected by measuring the signal characteristic of the respective fluorescent dye, while simultaneously determining the relative position of the individual fluorescence signals on the reaction surface
• e) to generate unlabelled (NTs or) NSKFs, cleave the linker component and the label moiety from the nucleated components attached to the complementary strand;
• f) washing the stationary phase obtained in step e) under conditions suitable for removing the marker component;

if necessary, the steps a) to f) are repeated several times,
whereby the relative position of individual NSKF primer complexes on the reaction surface and the sequence of these NSKFs are determined by specific assignment of the fluorescence signals to the nuc macromolecules detected in step d) in successive cycles at the respective positions.

One Further additional aspect 54 of the invention relates to a method according to Additional aspect 53, characterized in that the steps a) to f) repeated the cyclic synthesis reaction several times, wherein one uses only one type of nuc-macromolecule in each cycle.

One Further additional aspect 55 of the invention relates to a method according to Additional aspect 53, characterized in that the steps a) to f) repeated the cyclic synthesis reaction several times, wherein in each cycle two differently marked species the nuc macromolecules starts.

One Further additional aspect 56 of the invention relates to a method according to Additional aspect 53, characterized in that the steps a) to f) repeated the cyclic synthesis reaction several times, wherein in each cycle four differently marked species the nuc macromolecules starts.

One additional aspect 57 of the invention relates to a method according to Zusatzaspekt 53, characterized in that the NSKs variants a known reference sequence and the steps a) to f) of repeated cyclic synthesis reaction several times, where in the Cycles alternately two different marked species the nuc macromolecules and insert two unmarked NTs and one through the entire sequences Comparison with the reference sequence determined.

One Further additional aspect 58 of the invention relates to a method according to the additional aspects 53 to 57, characterized in that in the NSKFs each introduce a primer binding site (PBS), wherein one with double-stranded one NSKs on both complementary single strands each introduces a PBS and wherein the primer binding sites are the same for all NSKFs or have different sequences.

One Further additional aspect 59 of the invention relates to a method according to the additional aspects 53 to 57, characterized in that the NSKFs with primers in a solution under conditions that are favorable for the hybridization of Primers to the primer binding sites (PBSs) of the NSKFs are suitable, where the primers are mutually identical or different sequences and then the formed NSKF primer complexes on the reaction surface binds.

One Further additional aspect 60 of the invention relates to a method according to the additional aspects 53 to 57, characterized in that the NSKFs first on the reaction surface immobilized and only afterwards with primers under conditions that bring about hybridization the primer to the primer binding sites (PBSs) of the NSKFs suitable are, being NSKF primer complexes are formed, wherein the primers with each other the same or different Have sequences.

A further additional aspect 61 of the invention relates to a method according to additional aspects 53 to 60, wherein the incorporation reaction is carried out in parallel on 10 to 100,000 different sequence populations Further additional aspect 62 of the invention relates to a method according to additional aspects 53 to 60, carried out at the 100,000 to 100,000,000 different sequence populations, the incorporation reaction in parallel.

One Further additional aspect 63 of the invention relates to a method according to Additional aspects 28 to 62, wherein the sequences of nucleic acid chains be determined

One additional aspect 64 of the invention relates to a method according to Additional aspects 28 to 63, where the marker component is fluorescent is marked

2. 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 enzymatischer Einbau von markierten Nukleotiden an den 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 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 enzymatic incorporation of labeled nucleotides on 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. Of the Contact between labeled components (both conventionally modified Nucleotides as well as nuc-macromolecules) and the surface the solid phase leads to their unspecific binding to the surface. This non-specific binding from labeled constituents to the surface constitutes an important source for the Background signal.

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 microns 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 (for example 100 cycles). 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.

It have already been several variants of the surface preparation or process design suggested that to a reduced background signal through lead to non-specific binding of labeled components.

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. In one embodiment, labeled components are marked with conventional markers, such as conventional ones labeled nucleotides. Such nucleotides can be used, for example, 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).

In another embodiment labeled components are nuc-macromolecules.

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 conventionally modified nucleotides (ribonucleoside triphosphates, 2'-deoxyribonucleoside 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, 5.403, 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 conventionally-modified 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. 6,254,575, 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,
Nuc-macromolecules are shown in Example 8.

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 is in one of the following ranges (indication: number of nucleic acid strands per 100 μm ² ): 0.1-1 / 100 μm ² , 1-10 / 100 μm 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 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 μm 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 J 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 .

In a further embodiment, the density of the extendable primer-template complexes lies in one or more of the following ranges (indication: number of primer template complexes per 100 μm ² that are capable of extension): 0.1-1 / 100 μm ² , 1-10 / 100 μm 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 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 μm 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 , 10-100 / 100 μm ² , 50-200 / 100 μm ² , 50-500 / 100 μm ² .

In a random distribution of primer-template complexes on the surface, a certain proportion of the population can be distinguished as single sequencing sites from the adjacent sequencing sites. The size of the portion of the population in which signals from incorporated nucleotides on primer-matri zenkomplexen can be individually recognized, ie without overlap with the signals from the adjacent sites, depends essentially on the density of the fixed and Extensible primer template complexes and the resolution of the detection apparatus. Examples of calculation of proportions of primer-template complexes detected without overlap with the adjacent sites are given in Tables 1-8. In this embodiment, only extension-capable primer template complexes are considered.

you sees clearly that the relative share of individually recognized Primer Matrizekomplexe at a low density of primer-template complexes on the surface is large. With increasing density or decreasing resolution decreases the relative proportion the individually recognized events on primer-template complexes, as always more places overlap with the neighboring places and from the apparatus do not distinguish as events on individual primer template complexes to let.

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 μm, the preferred actual density is between 40 and 1300 sites per 100 μm ² , in another embodiment the preferred density is between 100 and 600 sites per 100 μm 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 100μm ² , in another embodiment, the preferred density is between 70 and 300 sites per 100μm 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.

• 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 enzymatischer Einbau von markierten Nukleotiden an den 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

The surface of the invention is used in highly parallel sequencing of individual nucleic acid chain molecules. This method essentially 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 enzymatic incorporation of labeled nucleotides on 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

• 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.It is possible to use both conventionally modified nucleotides and nuc-macromolecules. This method can be carried out in several embodiments, differing inter alia in the type of control of the cyclic Reaktkion:
An embodiment (A) of this method includes the following steps:
Fragments (NSKFs) of single-stranded NSKs about 50 to 1000 nucleotides in length can be generated, which can be overlapping partial sequences of the total sequences
the NSKFs are bound in a random array using one or more different primers in the form of NSKF primer complexes on the 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 from each other by measuring different fluorescence signals, 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) incubating the stationary phase obtained in step a) under conditions suitable for extending the complementary strands, wherein the complementary strands are each extended by one NT * the, man
• 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 unlabelled (NTs or) NSKFs, the fluorescent dyes and the sterically demanding ligands of the cleaved at the complementary strand NTs cleaved, one
• f) washing the stationary phase obtained in step e) under conditions suitable for removing the fluorescent dyes and the ligands;

if necessary, the steps a) to f) are repeated several times,
whereby the relative position of individual NSKF primer complexes on the reaction surface and the sequence of these NSKFs are determined by specific assignment of the fluorescence signals to the NTs detected in step d) in successive cycles at the respective positions.

Examples for nucleotides, which bear a steric hindrance are in Examples 7 A to Pictured 7D.

• 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 3'-OH-Nukleotidposition 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 und die strukturelle Modifikation an der 3'-OH-Nukleotidposition abspaltbar sind, 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 strukturelle Modifikation an der 3'-OH-Nukleotidposition 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.Another embodiment (B) of this method includes the following steps:
Fragments (NSKFs) of single-stranded NSKs about 50 to 1000 nucleotides in length can be generated, which can be overlapping partial sequences of the total sequences
the NSKFs are bound in a random array using one or more different primers in the form of NSKF primer complexes on the 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, wherein the simultaneous use of at least two NTs fluorescence dyes present on the NTs * are selected such that the NTs used can be distinguished from one another by measuring different fluorescence signals, wherein the NTs * are structurally modified at the 3'-OH nucleotide position such 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 and the structural modification at the 3'-OH nucleotide position are cleavable, 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) to generate unlabelled (NTs or) NSKFs which cleave the fluorescent dyes and the structural modification at the 3'-OH nucleotide position of the NTs attached to the complementary strand, one
• f) washing the stationary phase obtained in step e) under conditions suitable for removing the fluorescent dyes and the ligands;

if necessary, the steps a) to f) are repeated several times,
whereby the relative position of individual NSKF primer complexes on the reaction surface and the sequence of these NSKFs are determined by specific assignment of the fluorescence signals to the NTs detected in step d) in successive cycles at the respective positions.

Examples of nucleotides which have structural modification at the 3'-OH nucleotide position are described in (Odedra WO 0192284, Odedra WO 03048178, 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 , Ju et al. WO 02/29003, Milton et al. WO 2004/018493, Milton et al. WO 2004/018497).

• a) zu den an die Oberfläche gebundenen NSKF-Primer-Komplexen eine Lösung zugibt, die eine oder mehrere Polymerasen enthält, wobei die Polymerasen an die NSKF-Primer-Komplexe binden, man
• b) die erhaltene Oberfläche unter Bedingungen wäscht, die zur Entfernung von nicht gebundenen Polymasen geeignet sind, man
• c) zu den an die erhaltene Oberfläche gebundenen NSKF-Primer-Polymerase-Komplexen eine Lösung mit modifizierten Nukleotiden zugibt, wobei Nukleotide 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 der Fluoreszenzfarbstoff abspaltbar ist, man
• d) 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
• e) die in Stufe b) erhaltene stationäre Phase unter Bedingungen wäscht, die zur Entfernung nicht in einen komplementären Strang eingebauter NTs* geeignet sind, man
• f) 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
• g) zur Erzeugung unmarkierter (NTs oder) NSKFs die Fluoreszenzfarbstoffe von den am komplementären Strang angefügten NTs* abspaltet, man
• h) die in Stufe e) erhaltene stationäre Phase unter Bedingungen wäscht, die zur Entfernung der Fluoreszenzfarbstoffe 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.Another embodiment (C) of this method includes the following steps:
Fragments (NSKFs) of single-stranded NSKs about 50 to 1000 nucleotides in length can be generated, which can be overlapping partial sequences of the total sequences
the NSKFs are bound in a random array using one or more different primers in the form of NSKF primer complexes on the 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, the polymerases binding to the NSKF primer complexes;
• b) washing the resulting surface under conditions suitable for removing unbound polymases;
• c) adding to the resulting surface bound NSKF primer polymerase complexes a solution with modified nucleotides, wherein nucleotides are labeled with fluorescent dyes, wherein the simultaneous use of at least two NTs * each located on the NTs * fluorescent dyes so selected are that the NTs used can * differ by measuring different fluorescence signals from each other, the fluorescent dye is cleavable, one
• d) the stationary phase obtained in step a) is incubated under conditions suitable for extending the complementary strands, wherein the complementary strands are each extended by one NT *
• e) washing the stationary phase obtained in stage b) under conditions which are suitable for the removal of non-complementary NTs *
• f) the individual NTs * incorporated into complementary strands are detected by measuring the signal characteristic of the respective fluorescent dye, while at the same time determining the relative position of the individual fluorescence signals on the reaction surface
• g) for the production of unlabelled (NTs or) NSKFs, the fluorescent dyes from the attached to the complementary strand NTs * cleaved, one
• h) washing the stationary phase obtained in step e) under conditions suitable for removing the fluorescent dyes;

if necessary, the steps a) to f) are repeated several times,
whereby the relative position of individual NSKF primer complexes on the reaction surface and the sequence of these NSKFs are determined by specific assignment of the fluorescence signals to the NTs detected in step d) in successive cycles at the respective positions.

In this embodiment can both processive polymerases such as Sequenase 2 or Klenow fragment Exo minus as well as destructive polymerases, for example Polymerase Beta, to be used. In a preferred embodiment be in this embodiment destructive polymerases used, for example, polymerase beta ("Animal Cell DNA Polymerases "1983, Fry M., CRC Press Inc., commercially available from Chimerx). After this Incorporation of a single nucleotide dissolves the polymerase of the primer-template complexes and thus is not able to incorporate another labeled nucleotide.

The Binding of polymerases to the primer-template complexes is reversible. After a certain time the polymerase leaves the polymerase primer-template complexes. This happened with a Half-life T. It is advantageous to carry out steps (b) to (d) in this embodiment the notification within fractions of the half-life (T) to to perform a half-life (T). When the half-life the polymerase primer-template complexes for example, 1 sec, For example, steps (b) through (d) should occur within one second. For longer Habit times can according to the times for adapted steps b to d become. For a quick exchange of solutions in reaction vessels many examples are known, e.g. Stop-flow apparatus.

• 1) Nukleotide mit einem kurzen Linker: a) mit einem sterischen Hindernis (wie Nukleotide in Beispielen 7A-D), b) mit einer an die 3'-Nukleotidposition gekoppelten Gruppe, wie in (Odedra WO 0192284, Odedra WO 03048178, Canard US Pat. Nr. 5798210, Kwiatkowski US Pat. Nr. 6255475, Kwiatkowski WO 01/25247, Parce WO 0050642, Tsien WO 9106678, Faulstich et al. DE 4418691 , Ju et al. WO 02/29003, Milton et al. WO 2004/018493, Milton et al. WO 2004/018497) dargestellt, dabei der Marker über einen Linker sowohl an der Base gebunden sein, als auch an der 3'-Gruppe selbst. Die 3'-OH-Gruppe kann Modifikationen tragen, die leicht abspaltbar sind..
• 2) Nukleotide mit einem langen Linker, wie Beispiel 7E angegeben. Solche Nukleotide haben mehrere Vorteile den mit einem kurzen Linker: a) Durch einen langen Linker ist die sterische Beeinflußung der Polymerase durch den Farbstoff sehr gering, b) nach den Einbau eines solchen Nukleotides liegt der Marker (in der Regel ein Fluoreszenzfarbstoff) in einem relativ großen Abstand vom Doppelstrang, so dass die Fluoreszenzeigenschaften, wie Quantumausbeute, Spektrum, Quenching usw. von der Basenzusammensetzung weniger abhängig sind als bei Nukleotiden mit einem kurzen Linker. Der Linker kann an der Base oder am Zucker gebunden sein.

The nucleotides that can be used in this embodiment (C) can have different structures. The modifications affect both the position of the modification (base or sugar) and the length of the linker that lies between the label and the nucleotide.

• 1) nucleotides with a short linker: a) with a steric barrier (such as nucleotides in Examples 7A-D), b) with a group linked to the 3'-nucleotide position, as in (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, Tsien WO 9106678, Faulstich et al. DE 4418691 , Ju et al. WO 02/29003, Milton et al. WO 2004/018493, Milton et al. WO 2004/018497), the marker being bound via a linker both to the base and to the 3'-group itself. The 3'-OH group can carry modifications which can be easily cleaved off.
• 2) nucleotides with a long linker, as given in example 7E. Such nucleotides have several advantages with a short linker: a) A long linker is the steric influence of the polyme rase through the dye is very low, b) after incorporation of such a nucleotide, the marker (usually a fluorescent dye) is at a relatively large distance from the duplex, so that the fluorescence properties, such as quantum yield, spectrum, quenching, etc., of the base composition are less are dependent on nucleotides with a short linker. The linker may be attached to the base or to the sugar.

If in this embodiment Nucleotide modifications are used, which except dye an additional Wear modification, so can additional steps in this embodiment For example, removal of the modification can be done parallel to step (g) or after this step.

• a) zu den an die Oberfläche gebundenen NSKF-Primer-Komplexen eine Lösung zugibt, die eine oder mehrere Polymerasen und nur eine Art der vier modifizierten Nukleotide (NTs*) enthält, die mit Fluoreszenzfarbstoffen markiert sind.
• 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 oder mehrere 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 und der Signalintensität detektiert, wobei man gleichzeitig die relative Position der einzelnen Fluoreszenzsignale auf der Reaktionsoberfläche bestimmt, man
• e) zur Erzeugung unmarkierter (NTs oder) NSKFs die Fluoreszenzfarbstoffe 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 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.Another embodiment (D) of this method includes the following steps:
Fragments (NSKFs) of single-stranded NSKs about 50 to 1000 nucleotides in length can be generated, which can be overlapping partial sequences of the total sequences
the NSKFs are bound in a random array using one or more different primers in the form of NSKF primer complexes on the 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 only one type of the four modified nucleotides (NTs *) labeled with fluorescent dyes.
• 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 or more NT *, respectively
• 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 and the signal intensity, while simultaneously determining the relative position of the individual fluorescence signals on the reaction surface
• e) for the production of unlabelled (NTs or) NSKFs, the fluorescent dyes from the attached to the complementary strand NTs * cleaved, one
• f) washing the stationary phase obtained in step e) under conditions suitable for removing the fluorescent dyes;

if necessary, the steps a) to f) are repeated several times,
whereby the relative position of individual NSKF primer complexes on the reaction surface and the sequence of these NSKFs are determined by specific assignment of the fluorescence signals to the NTs detected in step d) in successive cycles at the respective positions.

Examples for nucleotides, the one big one steric hindrance are in Examples 7 A to D, nucleotide with a lesser steric hindrance is shown in Example 7F.

polymerases differ in their ability modified nucleotides (Augustin et al., Progress toward single-molecule sequencing: enzymatic synthesis of nucleotide-specifically labeled DNA " Biotechnol 2001, v. 86, 289-301). In this embodiment of the process, polymers can be used, the several modified nucleotides in succession Install. Nucleotides with a long linker, such as in Example 7E, since in these nucleotides a lower probability the influence the fluorescence property consists of the adjacent incorporated nucleotide molecules.

The in the process according to the invention determined partial sequences preferably have a length of 10 up to 100 nucleotides.

Vorzusweise 20 to 200 steps are carried out incorporating nucleotides become.

The above-mentioned embodiments of the method may include additional additional steps. These steps may be periodic in every cycle or intermittently between cycles. In one embodiment of the process, these steps serve to modify reactive groups after cleavage of fluorophores (see Example 6.1). In another embodiment of the method, such steps serve to remove certain reagents from the surface, for example polymerases bound to the nucleic acid strands. This can be done, for example, by treating the solid phase with a buffer solution capable of complexing Mg 2+ (eg, EDTA buffer solution), or by treating the solid phase with a solution of high salt concentration. Another possibility for removing the proteins bound to the nucleic acid chains is a protease treatment.

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 5.21, 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 repeating steps a) to f) of the cyclic synthesis reaction in the embodiments (A) and (B) and the steps a) to h) in the embodiment (C) several times , where one

• 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.

in the Another is the embodiment (A) of the method is considered in more detail for clarity, wherein This should not be a limitation lead the inventive method.

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.

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, marked NTs * are incorporated into the newly synthesized strand. The polymerase incorporates only a single labeled NT * into the growing chain. This is achieved by the reversible coupling of a terminating sterically demanding group to the NTs *. The installation of another marked NT * is thereby made impossible. The The sterically demanding 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 primer 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" 5. 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 5.4992, 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.

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 5.8794, "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, of a single primer binding site and a hybridized primer (bound NSKF-primer complexes) to obtain. In detail, you 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 the nucleic acid chain fragments (NSKFs) can be carried out by a plurality of methods, for example by fragmentation of the starting material with ultrasound or by endonucleases asen ("Molecular Cloning" 1989 J. Sambrook et al., Cold Spring Harbor Laboratory Press), such as by unspecific endonuclease mixtures. According to the invention, the ultrasonic fragmentation is preferred. You can set the conditions so that fragments with an average length of 100 bp to 1 kb arise. These fragments are then filled at their ends by the Klenow fragment (E. coli polymerase I) or 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, 5,231, Ledbetter et al., J. Biol. Chem., 1994 v.269 p.31544, Kolls et al. Anal. 1993 v.208 5.264, decraene et al. Biotechniques 1999 v.27 S.962).

Introduction of a primer binding site into 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 380 A Applied Biosystems can be used. Oligonucleotides of a particular composition with or without modifications but 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 NSKFs subsequently hybridized to the bound primers 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.

Immobilization of NSKFs to the surface may therefore be by direct or indirect binding respectively.

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, 5.3678, 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

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 as a substrate (e.g., cDNA) are useful as polymerases in principle 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), Klenow fragment of DNA polymerase I without 3'-5 'exonuclease activity (Amersham Pharmacia Biotech), polymerase beta of various origins (Animal Cell DNA Polymerases "1983, Fry M., CRC Press Inc., commercially available from Chimerx) thermostable Polymerases such as Taq polymerase (GibcoBRL), proHA DNA polymerase (Eurogentec), Vent exo minus, Pwo polymer. DNA-dependent RNA polymerases can also be used be, e.g. 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.

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) Es ist vorteilhaft, wenn die Reaktion so gesteuert wird, dass die Polymerase NT*s einzeln einbaut (Kontrolle der Reaktion).
• 2) NT*s müssen eine Markierung tragen, der den Anforderungen der Detektion genügt.
• 3) Die Markierung 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) It is advantageous if the reaction is controlled so that the polymerase incorporates NT * s individually (control of the reaction).
• 2) NT * s must bear a mark that meets the requirements of detection.
• 3) The label 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.

The label can be low molecular weight (eg conventionally modified nucleotides) or macro molecular (eg nuc-macromolecules).

Conventionally modified nucleotides include several types:
One type of nucleotide that can be used in the method of the invention carries a sterically bulky group that is coupled to the base. The sterically demanding group exerts a strong steric influence on the reaction. Examples of such nucleotides are modifications given under Examples 7A-D.
Another type of nucleotide that can be used in the method of the invention is modified at the 3'-OH nucleotide position. Examples of these nucleotides are described in Odedra WO 0192284, Odedra WO 03048178, Herrlein et al. Helvetica Chimica Acta, 1994, V. 77, p. 586, Canard US. Pat. No. 5798210, Kwiatkowski US Pat. No. 6255475, Kwiatkowski WO 01/25247, Parce WO 0050642.
Another type of nucleotides that can be used in the method of the invention is modified at the 3'-OH nucleotide position and is reversibly labeled at the base. Examples of such nucleotides are described in Milton et al. WO 2004/018493, Milton et al. WO 2004/018497,
Another type of nucleotides that can be used in the method according to the invention, carries a label which is coupled via a long linker to the nucleotide, s. Example 7E. This modification exerts a weak steric influence on the reaction.

Nuc-macromolecules are shown in Example 8.

in the The following are conventionally modified nucleotides with a sterically demanding group.

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 may be preferred to install a single NT *. The installation of a next NT * is sterically inhibited. These NTs * thus appear as terminators of the synthesis. To the removal of the sterically demanding group can incorporate the next complementary NT * become.

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 the sterically demanding group

fluorescent dyes represent examples of 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, 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 that the installation of the marked NT * to which it is attached, does not bother significantly and no irreversible disorder causes 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, Plenum 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, said compound belongs to chemically or enzymatically cleavable or photolabile compounds. As examples of chemically cleavable groups are ester, thioester and disulfide Ver Shan S. Wong 1993 CRC Press Inc., Herman et al., Methodology in Enzymology 1990 v.184 p.584, Lomant et al., J. Mol. Biol., 1976. v.104 243, "Chemistry of carboxylic acids and esters" S.Patai 1969 Interscience Publ.) Examples of photolabile compounds can be found in the 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, PhD Thesis "New Photolabile Protecting Groups for Light-Controlled Oligonucleotide Synthesis" H.Giegrich, 1996, Konstanz, Dissertation "New Photolabile Protecting Groups for Light-Controlled Oligonucleotide Synthesis" SMBühler, 1999, Constance) ..

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 Splitting is going on preferably chemically (e.g., in mild acidic or basic environment for one Ester compound or by adding a reducing agent, e.g. Dithiothreitol or mercaptoethanol (Sigma) in the cleavage of a Disulfide compound), or physically (e.g., by illumination the surface with light of a certain wavelength for the cleavage of a photolabile Group, Dissertation "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.

Exchange of reagents with the progress of sequencing:

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 replaced. For example, at the beginning a sequencing reaction nucleotide modifications with a short linker used, for example Example 7C or 7D. In the course of sequencing then nucleotide analogues are used with longer linkers, for example, Example 7A or 7E. At the end of the sequencing can then also nucleotides with an even longer one Linker can be used, for example Example 7B. In one embodiment Of the method, it is advantageous the first 25% of the corresponding Perform cycles with a nucleotide analog, the next 25% of the corresponding Cycles with another, etc. The exchange of nucleotides can be performed in a different sequence, for example, after 10% of all cycles, after 20%, etc. thereby nucleotides with, for example always 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 single-molecule 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.

Use of the surface:

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, 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 forms part of a reagent vessel, a Micro channel device or an array of reagent vessels, for example a microtiter plate, and the surface is made directly in such a reagent vessel or microchannel. different design options for microfluidic channels known to those skilled in the art (e.g., "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 preparation of a surface for the methods for highly parallel sequencing of individual nucleic acid chains essentially involves a combination of a removal step of the outer layer of the solid phase by a chemical or physical action and a coupling of nucleic acids to 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, ISBN0-306-43473-3, "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 a further embodiment can Linker molecules 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 groups ("Silane Coupling Agents" 2nd ed. 1991, ISBN0-306-43473-3, "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,).

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 the person skilled in the art ("Silane Coupling Agents" 2 Ed. 1991, ISBN0-306-43473-3, "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. 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.

3. Examples:

Examples should serve to illustrate the invention and not to limit. First, the Preparation of the solid phase, then be examples for Sequencing indicated.

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: Parafilm (Merck, Darm city) 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).

at further description of the processing of the surface will be provided that coarse contaminants from the surface of the solid phase have been removed. Also other both chemical, physical as well as mechanical steps may precede the production of the surface according to the invention, with the restriction, 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. 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.

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 ul of a 50 .mu.mol / l solution of dUTP-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.

Results:

• Behandlung der Oberfläche: wässrige HF-Lösung (v/v %)
• Spalte A) Ergebnis des Tests 1.1: dCTP-Cy3, 1μmol/l, 5' RT, Mittelwert der gemessenen Signale auf einer Fläche von 100μm²,
• Spalte B) Ergebnis des Tests 1.2: dUTP-AA-SS-Cy3, 1 μmol/l, 5' RT, Mittelwert der gemessenen Signale auf einer Fläche von 100μm²,
• Spalte C) Ergebnis des Tests 1.3: Oligo-T7-19-Cy3, 1 μmol/l, 5' RT, Mittelwert der gemessenen Signale auf einer Fläche von 100μm².
• Spalte D) Ergebnis des Tests 1.3: Oligo-B1-Cy3, 1 μmol/l, 5' RT, Mittelwert der gemessenen Signale auf einer Fläche von 100μm².
• Spalte E) Ergebnis des Tests 1.3: Oligo-F1-Cy3, 1 μmol/l, 5' RT, Mittelwert der gemessenen Signale auf einer Fläche von 100μm².
• Spalte F) Ergebnis des Tests 1.3: Oligo-T40-Cy3, 1 μmol/l, 5' RT, Mittelwert der gemessenen Signale auf einer Fläche von 100μm².
• Spalte G) Ergebnis des Tests 1.3: Oligo-dA26-Cy3, 1 μmol/l, 5' RT, Mittelwert der gemessenen Signale auf einer Fläche von 100μm².
• Spalte H) Ergebnis des Tests 1.3: Oligo-2-TMR, 1 μmol/l, 5' RT, Mittelwert der gemessenen Signale auf einer Fläche von 100μm².
• Spalte I) Ergebnis des Tests 1.4: dUTP-Cy3, 50 μmol/l, 5' RT, Mittelwert der gemessenen Signale auf einer Fläche von 100μm².

The data for binding of labeled substances to the surface are shown in Table 9. Table 9:

• Surface treatment: 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, average of the measured signals over an area of 100μm 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

• Spalte A) wässrige HF-Lösung (v/v %)
• Spalte B) dCTP-Cy3, 1μmol/l, 5' RT, Mittelwert der gemessenen Signale auf einer Fläche von 100μm².
• Spalte C) Oligo-T7-19-Cy3, 1 μmol/l, 5' RT, Mittelwert der gemessenen Signale auf einer Fläche von 100μm².

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. Table 10 shows results of test 1.1 and 1.3 with oligo-T7-19-Cy3 after applying different concentrations of HF solution. 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 hours at RT 5M NaOH solution is replaced with a 50 mmol / l Tris-HCl buffer, pH 9.0, and the channel is washed five times with this buffer. The first control of the non-specific 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:

• Behandlung: 5 mol/l wässrige NaOH-Lösung
• Spalte A) dCTP-Cy3, 1μmol/l, 5' RT, Mittelwert der gemessenen Signale auf einer Fläche von 100μm².
• Spalte B) Oligo-T7-19-Cy3, 1 μmol/l, 5' RT, Mittelwert der gemessenen Signale auf einer Fläche von 100μm².

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.

A such density of unspecifically bound signals allows a good Differentiation of individual signals from single molecules at the Surface, s. Table 1 to 8 and leads to a lesser overlap with specific signals.

Example 2.2

• Spalte A) wässrige NaOH-Lösung, Angabe für mol/l
• Spalte B) dCTP-Cy3, 1μmol/l, 5' RT, Mittelwert der gemessenen Signale auf einer Fläche von 100μm².
• Spalte C) Oligo-T7-19-Cy3, 1 μmol/l, 5' RT, Mittelwert der gemessenen Signale auf einer Fläche von 100μm².

To illustrate the effect of the removal of the outer layer by the NaOH treatment, results of a titration series are presented below. The solid phase was treated with different concentrations of the NaOH solution. The titration procedure and further treatment were similar to those in Example 2.1. Table 12 shows results of test 1.1 and 1.3. Control of non-specific binding: Table 12

• Column A) aqueous NaOH solution, indication for mol / l
• 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 retain its characteristic of low non-specific binding of labeled substances for several months covered with a solution. 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 of the invention, the solid phase is regenerated with a Surface during all subsequent treatment steps not dried and at no time has a direct contact with air. The storage of the freshly regenerated surface takes place in contact with a solution, for example with water or another solution, which has been described above. All further modifying steps in the production take place through 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 the regenerated surfaces obtained under Example 1, 2 or 3 takes place for example with silylation reagents. Many examples of silica Modification are known ("silanes Coupling Agents "2. Ed. 1991, ISBN0-306-43473-3).

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 without drying as in follows processed:

• 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 can be done for example at one of the two ends of the chain or over a group coupled to one of the nucleotides within the chain is. The covalent coupling of nucleic acids can be bound to those on the surface Left or directly to the glass surface done. The affine bond For example, between a fixed on the surface Streptavidin and bound to the nucleic acid biotin done. Another example of the affine binding of nucleic acids represents the hybridization of complementary nucleic acid chains to those on the surface fixed nucleic acids The following are examples of covalent and affine coupling of the nucleic acid chains to the surface shown.

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

• Spalte A) Ergebnis des Tests 1.1: dCTP-Cy3, 1μmol/l, 5' RT, Mittelwert der gemessenen Signale auf einer Fläche von 100μm²,
• Spalte B) Ergebnis des Tests 1.2: dUTP-AA-SS-Cy3, 1 μmol/l, 5' RT, Mittelwert der gemessenen Signale auf einer Fläche von 100μm²,
• Spalte C) Ergebnis des Tests 1.3 (7A): Oligo-T7-19-Cy3, 1 μmol/l, 5' RT, Mittelwert der gemessenen Signale auf einer Fläche von 100μm².
• Spalte D) Ergebnis des Tests 1.4: dUTP-Cy3, 50 μmol/l, 5' RT, Mittelwert der gemessenen Signale auf einer Fläche von 100μm².

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, 1μmol / l, 5 'RT, average of measured signals over an area of 100μm 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

• Spalte A) Ergebnis des Tests 1.1: dCTP-Cy3, 1μmol/l, 5' RT, Mittelwert der gemessenen Signale auf einer Fläche von 100μm²,
• Spalte B) Ergebnis des Tests 1.2: dUTP-AA-SS-Cy3, 1 μmol/l, 5' RT, Mittelwert der gemessenen Signale auf einer Fläche von 100μm²,
• Spalte C) Ergebnis des Tests 1.3 (8A): Oligo-T7-19-Cy3, 1 μmol/l, 5' RT, Mittelwert der gemessenen Signale auf einer Fläche von 100μm².
• Spalte D) Ergebnis des Tests 1.4: dUTP-Cy3, 50 μmol/l, 5' RT, Mittelwert der gemessenen Signale auf einer Fläche von 100μm².

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 be influenced by the time, concentration of the reagents and temperature. Instead of a trimethoxysilyl group, another silylation reaction coupled to the oligonucleotide may also be used. Oligonucleotides with activated silyl groups can be diluted in organic solvents such as DMAA, DMF, DMSO or acetonitrile. It is also possible to use aqueous mixtures. The time of the modification can be between a few Minutes and several hours. The choice of a corresponding active silyl group depends on compatibility with reagents that come in contact with surface in later steps.

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 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-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

Here will be an example of the inventive method represented, in which the surface according to the invention can be used.

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, a highly parallel sequencing reaction is performed on an oligonucleotide. The following components were used:
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)
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: 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 of the aliquot of Klenow exo minus polymerase (Amersham-Bioscience, 750 Unit Klenow Exo minus) was added iodoacetamide (Sigma-Aldrich) to a concentration of 20 mmol / l and at RT stirred for 30 min. Further notes for the modification are given in Appendix 1:
An oligo-31 was hybridized to the anchor oligonucleotides:
A 100 nmol / l solution of Oligo 31 in the incorporation buffer (20 μl) was brought into contact with the surface of the solid phase at RT for 5 min, then the surface was washed with the incorporation buffer (100 μl). Subsequently, primer T7-19 was hybridized to the primer binding site in oligo 31:
A 10 nmol / L solution of T7-19 in the incorporation buffer (20 μl) was brought into contact with the surface of the solid phase for 5 min at RT, then the surface was washed with the incorporation buffer (100 μl).

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.

• 1) die Oberfläche der festen Phase mit den fixierten Primer-Matrizekomplexen wurde mit Spaltungspuffer (10 μl) 5 min bei RT inkubiert, anschließend mit dem Waschpuffer gewaschen, dann für 5 min bei RT mit Alkylierungspuffer (20 μl) gewaschen (zur Blockade von freien SH-Gruppen) und dann mit Waschpuffer gewaschen.
• 2) es wurde der Lösung "A,G" (10 μl) auf die Oberfläche gegeben und bei RT 5 min inkubiert, so dass gegebenenfalls eine Einbaureaktion von dATP und oder dGTP stattfinden kann. Anschließend wurde die Oberfläche mit dem Waschpuffer gewaschen.
• 3) Detektion

Cycle (A) includes the following steps:

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

• 1) die Oberfläche der festen Phase mit den fixierten Primer-Matrizekomplexen wurde mit Lösung "U" 5 min bei RT inkubiert und anschließend mit dem Waschpuffer gewaschen.
• 2) Detektion

Cycle (U) includes the following steps:

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

• 1) die Oberfläche der festen Phase mit den fixierten Primer-Matrizekomplexen wurde mit Lösung "C" 5 min bei RT inkubiert und anschließend mit dem Waschpuffer gewaschen.
• 2) Detektion

Cycle (C) includes the following steps:

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

Table 15

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 sequence to be determined "--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 present invention, the concentration of oligo-31 bound to the solid phase via the linker was determined by Half reduced. 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

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 with a free 3 'group on the surface fixed to the solid phase and serve as primers for the extension reaction can. Fragments of the M-13 plasmid were hybridized to these oligonucleotides, which were provided with a poly-dT route 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 the individual:

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.

• 1) Hybridisierung von Fragmenten von M-13-Plasmid
• 2) Detektion
• 3) Bleichen der Signale von den eingebauten markierten Nukleotiden.

Cycle (T) Includes the following steps:

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

• 1) die Oberfläche der festen Phase mit den fixierten Primer-Matrizekomplexen wurde mit Spaltungspuffer (10 μl) 5 min bei RT inkubiert und anschließend mit dem Waschpuffer gewaschen, anschließend Block-Puffer für 5 min bei RT behandelt.
• 2) es wurde die Lösung "A,G" (10 μl) auf die Oberfläche gegeben und bei RT 5 min inkubiert, so dass gegebenenfalls eine Einbaureaktion von dATP und oder dGTP stattfinden kann. Anschließend wurde die Oberfläche mit dem Waschpuffer gewaschen.
• 3) Detektion

Cycle (A) includes the following steps:

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

• 1) die Oberfläche der festen Phase mit den fixierten Primer-Matrizekomplexen wurde mit Spaltungspuffer (10 μl) 5 min bei RT inkubiert und anschließend mit dem Waschpuffer gewaschen, anschließend Block-Puffer für 5 min bei RT behandelt.
• 2) Detektion

Cycle (a) includes the following steps:

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

• 1) die Oberfläche der festen Phase mit den fixierten Primer-Matrizekomplexen wurde mit der Lösung "U" 5 min bei RT inkubiert und anschließend mit dem Waschpuffer gewaschen.
• 2) Detektion

Cycle (U) includes the 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 washed with the washing buffer.
• 2) detection

• 1) die Oberfläche der festen Phase mit den fixierten Primer-Matrizekomplexen wurde mit der Lösung "C" 5 min bei RT inkubiert und anschließend mit dem Waschpuffer gewaschen.
• 2) Detektion

(Abweichungen von diesen Schritten sind mit Kursiv angegeben)Cycle (C) includes the 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 washed with the washing buffer.
• 2) detection

(Deviations from 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

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 = ((N - A) × 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 mofifiziert 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, 7E: 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, 5-iodo-2'-deoxyuridine (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), diamine PEG (6kDa, Fluka).

Solvents were used, if necessary, in the absolute form (Fluka) or dried by standard methods. For solvent mixtures, the specified mixing ratios refer to the set volumes (v / v).

Example 7A:

Synthesis of dUTP-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 30mol / 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 min, NH ₄ HCO _{3 was} up to Concentration of 100 mmol / l was added and after a further 30 min 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-NHS, 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 l).

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 -CN ₂ -NH ₂ is separated from the excess mercaptoethanolamine on DEAE-cellulose in the borate buffer 10 mmol / l and eluted from the column with 0.3 mol / l NaCl.

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.

Example 7E:

Synthesis of dUTP-AA-SS-PEG dye, ( 14 ).

dUTP-AA-SS-PEG-Cy3,

First, SH-PEG-Cy3 was prepared. To 200 μl of a 10 mmol / l solution of diamine PEG (6kDa, Fluka) in 50 mmol / l borate buffer, pH 8, Cy3-NHS (Amersham-Bioscience) was added to a final concentration of 15 mmol / l. The reaction was carried out at RT for 30 minutes. Subsequently, NH ₂ -PEG-Cy3 was separated from dye residues by ultrafiltration with MWCO 3000, washed 3 times with 1 ml of 50 mmol / l borate buffer, pH 8, and in a volume of 200 μl 50 mmol / l borate buffer, pH 8, dissolved.

To this solution was added PDTP-NHS to final concentration of 50 mmol / L. The reaction was carried out at RT for 30 minutes. Subsequently, 50 μl of a 1 mol / l solution of NH ₄ HCO ₃ , pH 8, were added and the reaction mixture was incubated for a further 60 min. To reduce the disulfide bonds, 100 μl of a 1 mol / l TCEP solution, pH 8, was added to the reaction mixture. After 5 min at RT, the product of the reaction, the SH-PEG-Cy3, was separated from the low molecular weight components by ultrafiltration with MWCO 3000, washed 5 times with 1 ml of 50 mM Tris-HCl buffer, pH 7, and washed a volume of 200 .mu.l 50 mmol / l Tris-HCl, pH 7, dissolved. Immediately before coupling the nucleotide portion, the pH was adjusted to 9.0 with 1 mol / l NaOH.

Coupling of SH-PEG-Cy3 to the nucleotide part:

To 100μl 20 mmol / l dUTP-AA-PDTP in 50 mmol / l borate buffer, pH 9, was added to 100 μl of the solution with SH-PEG-Cy3 in 50 mmol / l Tris-HCl, pH 9.0. Reaction was at RT for 30 min carried out. The product of the reaction was purified by ultrafiltration with MWCO 3000 separated from low molecular weight components, 5 times with 1 ml of 50 mmol / l Tris-HCl buffer, pH 7, and washed in a volume of 200 μl 50 mmol / l Tris-HCl, pH 7, dissolved.

The dUTP-AA-SS-PEG-Cy3 obtained in this way can be isolated from DNA polymerases, for example Klenow fragment exo-minus or Taq polymerase, in the growing strand of nucleic acids are incorporated.

The contains dUTP-AA-SS-PEG-Cy3 a cleavable under mild conditions group, so that the linker can be cleaved with the dye from the nucleotide.

This Example shows a general possibility at Nukleotiden further modifications vorzunehnem. Further base-modified Nucleotide analogs, e.g. 5-propargyl-dCTP, 5-propargylamino-dUTP, 7-deaza-7-aminopropargyl-dGTP and 7-deaza-7-aminopropargyl-dATP can as stated above also be modified. Both ribonucleotides, 2'-deoxyribonucleotides and 2 ', 3'-deoxyribonuclides.

Example 8

Nuc-macromolecules for the sequencing method

General notes for the syntheses of nuc macromolecules

The Nuc-macromolecules according to the invention can be obtained different ways are synthesized. The chronological order The coupling steps can vary. For example, a first Linker component will be coupled to the Nuk component, then becomes coupled to the marker component.

on the other hand can one or more linkers are coupled to the marker component and subsequently the nuk component (s).

The Coupling between individual components of nuc-macromolecules can covalent or affine. Both chemical and enzymatic Coupling to the link individual components can be used. Couplings at amino and thiol groups serve as examples of covalent couplings (D. Jameson et al. Methods in Enzymology 1997, V. 278, 363, "The chemistry of the amino group "S. Patai, 1968, "The chemistry of the thiol group "S. Patai, 1974). Biotin-streptavidin binding or hybridization between complementary Nucleic acid strands or Antigen-antibody interactions provide examples of affine couplings.

The Macromolecular markers often offer a variety of coupling possibilities. A macromolecular marker can have multiple coupling sites for the linker have, for example, multiple binding sites for biotin, as in streptavidin the case is. A macromolecular marker can also contain several amino or thiol groups.

If nucleic acids can be used as macromolecular markers, they can have different sections for coupling other macromolecules.

At a macromolecular marker other macromolecules be bound, e.g. Enzymes.

One Nuc-macromolecule can carry macromolecular markers with different detection properties, For example, a nuc-macromolecule can have both multiple dye molecules as well Sites for affinity binding (e.g., by hybridization) to others macromolecules wear.

The Coupling between the nuc-component and the linker components is preferably covalent. Many examples of covalent coupling Nucleotides or their analogs are known (Jameson et al., Method in Enzymology, 1997, v. 278, p. 363-, Held et al. Nucleic acid research, 2002, v. 30 3857, Short US Pat. 6579704, Odedra WO 0192284). there For example, the coupling may be to phosphate, amino, hydroxy or Mercapto groups take place.

Often, the linker component can be built in several steps. For example, in the first step, a short linker having a reactive group is coupled to the nucleotide or nucleoside, eg, propargylamine linker to pyrimidines Hobbs et al. US Patent 5,047,519 or other linkers, eg, Klevan US Pat. 4,828,979, Seela US Pat. 6211158, US pat. 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 & nnucleic acids, 2003, v. 22, p. 391, Short US Pat. No. 6579704, Odedra WO 0192284, Herrlein et al. Helvetica Chimica Acta, 1994, V. 77, p. 586, Canard US. Pat. 5798210, Kwiatkowski US Pat. No. 6255475, Kwiatkowski WO 01/25247, Parce WO 0050642, Tsien WO 9106678, Faulstich et al. DE 4418691 , Odedra WO 0192284, Odedra WO 03048178, Milton et al. WO 2004/018493, Milton et al. WO 2004/018497, Dissertation "Synthesis of base-modified nucleoside triphosphates and their enzymatic polymerization to functionalized DNA", Oliver Thum, Bonn 2002. Some compounds can be purchased, for example from Trilink Biotechnologies, Eurogentec, Jena Bioscience.

These short linkers serve as coupling units L or their parts, and are part of the linker component in the final nuc-macromolecule.

in the second step may be the coupling of the nucleotide or nucleosides with a short linker to the linker polymer. polymers with reactive functional groups can be purchased (Fluka).

To the coupling of the nucleotide to the polymer can now be the marker component be paired as a last step.

It is often advantageous to couple a short linker to a nucleoside, then, if necessary, to convert this modified nucleoside into a nucleoside triphosphate (syntheses of triphosphates can be found, for example, in the following references: Held et al., Nucleosides, nucleotides & nucleic acids). 2003, v. 22, p. 391, Faulstich et al. DE 4418691 , T. Kovacs, L. Ötvös, Tetrahedron Letters, Vol. 29, 4525-4588 (1988) or dissertation "Synthesis of base-modified nucleoside triphosphates and their enzymatic polymerization to functionalized DNA", Oliver Thum, Bonn 2002).

Further Modifications can be performed on the nucleoside triphosphate analog.

precursors for modified Nucleosides can are for example available from Trilink Biotechnologies (San Diego, CA, USA) or at Chembiotech (Münster, Germany) commercial.

The Coupling between the linker component and the marker component for example, between reactive groups on the linker component and the marker component. Reagents for such couplings are described in "Chemistry of protein conjugation and cross-linking ", Wang, 1993, ISBN 0-8493-5886-8, in detail. The proceedings for handling and coupling of several macromolecules for different Types of macromolecules also shown in the above-mentioned patents. Further examples of couplings to and between the macromolecules are for proteins in "bioconjugation: protein coupling techniques for the biomedical sciences ", M. Aslam, 1996, ISBN 0-333-58375-2;" Reactive dyes in protein to enzyme technology ", D. Clonis, 1987, ISBN 0-333-34500-2; "Biophysical labeling methods in molecular biology "G. Likhtenshtein, 1993, 1993, ISBN 0-521-43132-8; "Techniques in protein modification" R. Lundblad, 1995, ISBN 0-8493-2606-0; "Chemical responsents for protein modification "R. Lundblad, 1991, ISBN 0-8493-5097-2; for nucleic acids in "Molecular Cloning", J. Sambrook, vol 1-3, 2001, ISBN 0-87969-576-5, for other types of polymers "Macromolecules, Chemical Structure and Synthesis ", Volume 1, 4, H. Elias, 1999, ISBN 3-527-29872-X.

There the marker component usually has many coupling sites, can made further modifications to complete nuc-macromolecules become. For example, excess amino groups be blocked or modified by further modifications.

ever by field of application and reaction conditions under which nuc macromolecules are used can, can different chemical bonding modes between individual parts the macromolecules be beneficial. So are suitable for example in methods with Steps with higher Temperatures, e.g. in hybridization or PCR, nuc macromolecules, the have covalent, thermostable bonds between individual parts.

following should exemplify some possibilities for the synthesis of nuc macromolecules being represented. These are not intended to limit the potential Synthetic pathways and not to limit the possible nuc-macromolecular structures.

In the following examples nuc-macromolecules are given with polyethylene glycol (PEG) as the linker component. Examples of the couplings of PEG to other molecules are in "poly (ethylene glycol): In particular, very different reactive groups can be used for the coupling: N-succinimidyl carbonate (US Pat. 5,281,698, US Pat. US 5,468,478 Amine (Buckman et al., Makromol Chem V.182, p.1379 (1981), Zalipsky et al., Eur. Polym., JV19, p. 1177 (1983)), succinimidyl propionate and succinimidyl butanoate (Olson et al in Poly (ethylene glycol) Chemistry & Biological Applications, 170-181, Harris & Zalipsky Eds., ACS, Washington, DC, 1997; U.S. Pat No. 5,672,662), succinimidyl succinate (Abuchowski et al., Cancer Biochem., Biophys. v. 7, p.175 (1984), Joppich et al., Makromol Chem 1v 80, p.1381 (1979), benzotriazole carbonate (US Pat. No. 5,650,234), glycidyl ether (Pitha et al., Eur. 94, p.11 (1979), Elling et al., Biotech, Appl. Biochem., V.13, p354 (1991), Oxycarbonylimidazoles (Beauchamp, et al., Anal., Biochem , 131, p. 25 (1983), Tondelli et al., J. Controlled Release v.1, p.251 (1985)), p-nitrophenyl carbonate (Veronese, et al., Appl. Biochem., Biotech. 11, 5.141 (1985); and Sartore et al., Appl. Biochem Biotech., V.27, p.45 (1991)), aldehydes (Harris et al., J. Polym. Sci., Chem. Ed. 22, p. 341 (1984), US. Pat. 5824784, US. Pat., 5252714), maleimides (Goodson et al., Bio / Technology v.8, p.343 (1990), Romani et al., Chemistry of Peptides and Proteins v.2, p.29 (1984)), and Kogan, Synthetic Comm. v.22, 5.2417 (1992)), orthopyridyl disulfides (Woghiren, et al., Bioconj. Chem. v. 4, p. 314 (1993)), acrylol (Sawhney et al., Macromolecules, v. 26, 5.581 (U.S. 1993)), vinylsulfones (US Patent No. 5,900,461). Further examples of couplings of PEG to other molecules are described in Roberts et al. Adv. Drug Deliv. Reviews v. 54, p. 459 (2002), US 2003124086 . US 2003143185 , WO03037385, US6541543 . US 2003158333 To find WO 0126692.

Other similar Polymers can in a similar way Art be coupled. Examples of such polymers are others Poly (alkylene glycols), copolymers of ethylene glycol and propylene glycol, Poly (olefinic alcohols), poly (vinylpyrrolidones), poly (hydroxyalkylmethacrylamides), Poly (hydroxyalkyl methacrylates), poly (saccharides), poly (x-hydroxy acids), poly (acrylic acid), poly (vinyl alcohol).

The Purification of the nuc-components of the nuc-macromolecules takes place by conventional means of nucleotide chemistry: for example with silica gel chromatography in a water-ethanol mixture, ion exchange chromatography in a salt gradient and reverse phase chromatography in a water-methanol gradient. For the Nucleotide purification optimized chromatography columns are e.g. offered by Sigma-Aldrich.

The Purification of macromolecular linker components and marker components can be performed by ultrafiltration, gel electrophoresis, gel filtration and dialysis take place, see "Bioconjugation: protein coupling techniques for the biomedical sciences ", M. Aslam, 1996, ISBN 0-333-58375-2.

The Mass of nuc macromolecules differs significantly from the mass of nucleotides. For this Reason, it is advantageous in the final purification steps ultrafiltration use. Therefore the nuc macromolecules only an average mass is enriched, ultrafiltration is suitable also as an analytical method for the separation of synthesis products.

to Characterization of nuc macromolecules can be different methods macromolecular chemistry, e.g. UV-Vis spectroscopy, Fluorescence measurement, mass spectroscopy, fractionation, size exclusion chromatography, Ultracentrifugation and electrophoretic techniques, such as IEF, denaturing and non-denaturing gel electrophoresis ("Macromolecules, Chemical Structure and Syntheses", Vol. 1, 4, H. Elias, 1999, ISBN 3-527-29872-X, "Bioconjugation: protein coupling techniques for the biomedical sciences ", M. Aslam, 1996, ISBN 0-333-58375-2).

The Measurement of free SH groups in a substance was done with Ellmans Reagent (5,5'-dithiobis (2-nitrobenzoic acid), Riddles et al. Method in enzyme. 1983, V.91, p. 49.

syntheses of modified nucleotides

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 be used (Sigma).

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.

The affinity isolation of nuc macromolecules can be used, for example, when nuc-macromolecules oligonucleotides Component of the marker component are. By hybridizing to a complementary, up A solid phase-fixed nucleic acid can be isolated selectively become.

The Determination of the yields for Dye-labeled products were made on UV-Vis spectrometers.

The completeness The coupling to strepavidin was monitored by a control titration a biotin dye (biotin-4-fluorescein, Sigma) 100 μmol / L in 50 mmol / l borate, pH 8, carried out at RT for 5 min. For a complete cast of biotin binding sites at strepavidin during In the synthesis, no labeling of streptavidin occurs. At a Insufficient reaction occurs binding to SA. analysis with UV-Vis.

Material:

Diamono PEG 10,000 (diamino-polyethylene glycol 10,000, Sigma), dUTP-AA (dUTP-allylamine, Jena-Bioscience, Jena, Germany (hereinafter referred to as Jena-Bioscience)), TTP (thymidine triphosphate, may also be characterized as dTTP, Sigma), 3'-amino-TTP (3'-amino-3'-deoxy-thymidine triphosphate Trilink Biotechnologies, Inc. of San Diego, CA, USA (hereinafter referred to as Trilink)), PDTP (3- (2-pyridinyl-dithio) -propionic acid), 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)), Cy3 (Dye, Amerscham Bioscience, Freiburg, Germany, hereinafter referred to 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) phosphine, Sigma), DTBP (3,3'-dithio-bis-propionic acid, Fluka), Biotin-NHS (biotin-N-hydroxysuccinimidyl ester, Sigma). J-Ac (iodoacetate, Sigma), iodoacetamide (Sigma), TEAE (tris- (2-aminoethyl) amines, Sigma), Maleimido-ES-NHS (maleimido-acetic acid N-hydroxysuccinimidyl ester, Sigma), EDA (ethylenediamine, Sigma), CDI (1,1'-carbonyldiimidazole, Sigma), NHS-PEG-maleimide, 3,400 Da, Biotin-PEG-NHS, 5,000 Da, mPEG-SPA 5,000 Da, mPEG-SPA 20,000 Da (Nectar Molecular Engineering, formerly Shearwater Corporation, Huntsville, AL, USA, (hereinafter referred to as nectar)), diamine PEG, 6,000 Da (Fluka), 3'-biotin-dT31 an oligonucleotide having a sequence of 31 thymidine monophosphates, with a biotin molecule at the 3'-end (MWG Biotech, Ebersberg near Munich, Germany, (hereinafter referred to as MWG Biotech)), 3'-SH-oligo-dT30 an oligonucleotide with a sequence of 30 thymidine monophosphates having a mercapto group at the 3'-end (MWG Biotech, Germany), 3'-amino-oligo-dT31-5'-Cy3 an oligonucleotide with a sequence of 31 thymidine monophosphates, with one over one Linker of 6 atoms at the 3'-end coupled amino group and a Cy3 dye coupled at the 5'-end (MWG Biotech, Germany), SA (streptavidin, Roche, Mannheim, Germany), SA-Cy2 (Cy2 dye-modified streptavidin, Amersham). QDot (Qdot 605 Streptavidin Conjugate, Quantum Dot, Hayward, Calif., USA, (hereinafter referred to as referred to as quantum dot). Poly-lysine 1000-2000 (poly-L-lysine hydrobromide 1000-2000 Da, Fluka), poly-lysine 10,000-20,000 (poly-L-lysine hydrobromide 10,000 -20,000 Da, Fluka).

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).

Synthesis of individual components:

Example 8.1:

dUTP-AA-PDTP, s. example 7A.

This example shows a general possibility to further modify nucleotides. Other base-modified nucleotide analogs such as 7-deaza-aminopropargyl guanosine triphosphate and 7-deaza-aminopropargyl adenosine triphosphates, 5-amino-propargyl uridine triphosphates may also be modified as indicated above. Both ribonucleotides, 2'-deoxyribonucleotides can be used, 18 to 21 ,

Example 8.2:

dCTP-PA PDTP,

The Synthesis was as for dUTP-AA-PDTP, Example 7A.

Example 8.3:

Biotin-PEG-ethyl-SH, 22

To 200 μl aqueous CA solution (100mmol / L), pH 8.5, adjusted with NaOH, was added 10 mg biotin-PEG-NHS added and at 40 ° C Stirred for 18 hours. Subsequently was 200 μl TCEP-solution (0.5 mol / l), pH 8.0, added and stirred for a further 10 min at RT. The Product was purified by ultrafiltration to 3,000 MWCO (Molecular Weight cut off) from other reagents.

The Product carries a reactive SH group that can be easily modified, for example under formation of disulfide bond.

Examples of coupling of linker components and marker components on nuc-components

Example 8.4:

dUTP-AA-PEG-biotin, 23

To 100μl aqueous solution dUTP-AA, 50mmol / L, pH 8.0, was given 10mg of Biotin-PEG-NHS and added 40 ° C 18 Hours stirred. Subsequently was the unreacted nucleotide by ultrafiltration, 3,000 MWCO, separated and the product of the reaction, the dUTP-AA-PEG-biotin, washed several times with water.

These Carries connection a nucleotide functionality and a macromolecular linker. Biotin serves as a coupling unit T. To this coupling unit T can macromolecular structures are coupled, e.g. streptavidin without that Substrate properties of these analogs are lost. This nuc macromolecule serves as a substrate for Polymerases.

biotin can also act as a low molecular weight marker unit with signal-mediating Function, which is coupled to the long linker.

The Product of the reaction is by definition an intermediate of a Nuc-macromolecule: the Linker length is significantly larger than 30 atoms and to the coupling unit T more macromolecules can be coupled become.

This example shows a general possibility to further modify nucleotides. Other base-modified nucleotide analogs such as 5-propargylamino-dCTP, 7-deaza-aminopropargyl-dGTP, 5-amino-propargyl-dUTP and 7-deaza-aminopropargyl-dATP may also be modified as noted above. Both ribonucleotides, 2'-deoxyribonucleotides can be used, 18 to 21 ,

The synthesis of this nucleotide serves as an example of the synthesis of precursors for the nuc-macro molecules.

Example 8.5:

dCTP-PA-PEG-maleimide,

The Synthesis was as for dUTP-AA-PEG-biotin, Example 8.4. As starting materials were dCTP-PA and maleimide-PEG-NHS.

These Compound has a nucleotide functionality and a macromolecular Linker. Coupling unit T at this linker is the maleimide group. Maleimide functionality can be macromolecular signal carrying molecules coupled carrying one or more SH groups.

Maleimide group can also act as a low molecular weight marker unit with signal-mediating Function, which is coupled to the long linker.

The Product of the reaction is by definition an intermediate of a Nuc-macromolecule: the Linker length is significantly larger than 30 atoms, the marker component is low molecular weight. At this marker component can macromolecular Structures are coupled without the substrate properties of this Analogues are lost.

This example shows a general possibility to further modify nucleotides. Other base-modified nucleotide analogs such as 5-allylamino-dUTP, 5-amino-propargyl-dUTP, 7-deaza-aminopropargyl-dGTP, and 7-deaza-aminopropargyl-dATP may also be modified as noted above. Both ribonucleotides, 2'-deoxyribonucleotides can be used, 18 to 21 ,

The Synthesis of this nucleotide serves as an example for the synthesis of precursors for the Nuc-macromolecules.

Example 8.6:

dUTP-AA-SS-PEG-biotin, 24 .

To 100μl, 10mmol / l Biotin-PEG-ethyl-SH in 50mM borate, pH 9.5, was 50μl 30mmol / l dUTP-AA-PDTP in 50mM borate, pH 9.5. Reaction was at RT for 18 hours touched. The separation is similar as for the synthesis of dUTP-AA-PEG-biotin, Example 8.4 described.

These Carries connection a nucleotide functionality and a macromolecular linker. Biotin serves as a coupling unit T. To this coupling unit T can macromolecular structures are coupled, e.g. streptavidin without that Substrate properties of these analogs are lost. This nuc macromolecule serves as a substrate for Polymerases. about Can streptavidin also other macromolecules, For example, enzymes or Nukleinsäurenge be coupled.

The Product of the reaction is by definition an intermediate of a Nuc Macromolecule: the linker length is significantly larger than 30 atoms.

biotin can also be used as signal-mediating low-molecular-weight marker unit be designated.

The Linker component can be used together with the marker component of the Nuk component under mild Conditions are split off. Such nucleotides can be found in Sequencing method be used.

Another example of the nuc macromolecules with a cleavable group under mild conditions: dUTP-AA-propionate-S-CO-PEG-biotin ( 25 ).

For this Reaction, the dUTP-AA-PDTP was first purified on RP-HPLC in water-methanol gradient.

To 10 μl of a 50 mmol / l solution of dUTP-AA-PDTP in water was added 20 μl of a 100 mmol / l solution from TCEP, pH 8. The reaction was carried out at RT for 10 min. The product of the reaction, dUTP-AA-propioate-SH, was separated from other reagents on silica gel preparative plates, LM 2. The product, dUTP-AA-Propionate-SH, remains on the starting line. It was eluted from the plate with water, concentrated and dissolved in 50 μl 50 mmol / l borate buffer, pH 8.

To this solution were 50 μl a freshly prepared 2% aqueous solution of biotin-PEG-NHS added. The reaction was carried out at RT for 30 min long. Subsequently was the product of the reaction, dUTP-AA-propionate-S-CO-PEG-biotin, by ultrafiltration with MWCO 3000 of low molecular weight reaction components separated, five washed with 0.5 ml of water and dissolved in a volume of 50 ul.

The thus, the dUTP-AA-propionate-S-CO-PEG-biotin was obtained may be of DNA polymerases, for example Klenow fragment Exo minus or Taq polymerase, incorporated into the growing strand of nucleic acids become.

At The biotin can be over that Straptavidin further marker components can be coupled.

The the dUTP-AA-propionate-S-CO-PEG-biotin contains one under mild conditions fissile group, so that the Linker with the dye can be cleaved from the nucleotide. Such nucleotides can be used in the sequencing method.

This example shows a general possibility to further modify nucleotides. Other base-modified nucleotide analogs such as 5-propargylamino-dCTP, 5-amino-propargyl-dUTP, 7-deaza-aminopropargyl-dGTP, and 7-deaza-aminopropargyl-dATP may also be modified as noted above. Both ribonucleotides, 2'-deoxyribonucleotides can be used, 18 to 21 ,

Couplings to marker components

Example 8.7:

(DUTP-16-biotin) 4-SA,

To 200μl one solution of biotin-16-dUTP 200μmol / l in 50 mM Tris-HCl, pH 8.0, 200 μl of a streptavidin solution, 1 mg / ml, in 50 mM Tris-HCl, pH 8.0. After 1 hour at RT was was the (dUTP-16-biotin) 4-SA from unreacted biotin-16-dUTP separated by ultrafiltration, 50,000 MWCO.

It a compound was obtained which has a nucleotide functionality and a carries macromolecular marker functionalities.

The Product of the reaction is by definition a conventionally modified Nucleotide: the linker length is less than 30 atoms, the marker component is macromolecular. It can be representative of conventional considered modified nucleotides with a macromolecular marker become.

These Compound is derived from polymerases (e.g., Klenow exo minus polymerase and terminal transferase) are not accepted as a substrate. The modification leads to Loss of substrate properties. Properties of biotin-streptavidin binding are e.g. in Gonzalez et al. Journal Biolog. Chem. 1997, v. 272 P. 11288.

Example 8.8:

(DUTP-16-biotin) 4-SA-Cy2,

The Coupling of dUTP-16-biotin to SA-Cy2 was as for (dUTP-16-biotin) 4-SA described carried out. The compound serves as an equivalent to compound from Example 8.7, wherein for visualization streptavidin fluorescently labeled.

Example 8.9:

(dUTP-AA-PEG-biotin) 4-SA-Cy2 and (dUTP-AA-PEG-biotin) 4-SA, 26

The Coupling of dUTP-AA-PEG-biotin to SA-Cy2 or to SA was described as for (dUTP-16-biotin) 4-SA carried out. To 200μl 1 μg / μl streptavidin were 10μl a solution of dUTP-AA-PEG-biotin, ca. 1 mmol / l, and stirred at RT for 1 h. After that the product was not ultrafiltered by 50,000 MWCO of that coupled dUTP-AA-PEG-biotin separated and the product 2 times washed with water.

One Part of the recovered (dUTP-AA-PEG-biotin) 4-SA-Cy2 was digested with Cy3-NHS modified: 50 μl (dUTP-AA-PEG-biotin) 4-SA-Cy2 were dissolved in 50 mmol / l borate, pH 8.5, up to the concentration of 1.4 μg / μl, and then with Cy3-NHS spiked. Final concentration of Cy3 was 10 mmol / l. The Reaction was carried out at RT for 1 h. The product, (dUTP-AA-PEG-biotin) 4-SA-Cy2 / Cy3, was separated by ultrafiltration with 30,000 MWCO.

Thereby became a nuc macromolecule prepared that has very few free amino groups on the marker part.

It a compound was obtained which has a nucleotide functionality, a long macromolecular linker and a macromolecular marker component wearing.

The The product of the reaction is by definition a nuc macromolecule: the linker length is significantly larger than 30 atoms, the marker component is macromolecular. It can be representative considered for nuc macromolecules become.

These Compound is derived from polymerases (e.g., Klenow exo minus polymerase and terminal transferase) as a substrate.

Example 8.10:

(dUTP-AA-PEG-biotin) 2- (dT31-TEG-biotin) 2-SA-Cy2 ,, 27

The coupling of dUTP-AA-PEG-biotin to SA-Cy2 was performed as described for (dUTP-16-biotin) 4-SA:
To 100 μl of a streptavidin-Cy2 solution, 20 μmol / l (1.2 mg / ml), in Tris-HCl, 50 mmol / l, pH 8, was added 80 μl 80 μmol / l dT31-3'-TEG-biotin (MWG Biotech) and Incubated at RT for 10 min. (TEG is a short linker between biotin and dT31). Thereafter, 100 μl of a solution of dUTP-AA-PEG-biotin 50 μmol / l in 50 mM Tris-HCl, pH 8.0, was added. After 10 min at RT, (dUTP-AA-PEG-biotin) 2- (dT31-TEG-biotin) 2-SA-Cy2 was purified by ultrafiltration, 50,000 MWCO.

The substance carries a nucleoside triphosphate functionality and a macromolecular marker functionalities (oligo-dT31). The oligo-dT31 consists of nucleoside monophosphates, which, however, do not participate in the enzymatic reaction and have only one signal-transmitting function. To such an oligonucleotide complementary nucleic acids can be hybridized, which have a signaling function ( 28 ). (general rules for the hybridization of nucleic acids are known in the art, Anderson "Nucleic Acid Hybridization", 1999).

The The product of the reaction is by definition a nuc macromolecule: the linker length is significantly larger than 30 atoms, the marker component is macromolecular. This connection is derived from polymerases (e.g., Klenow exo minus polymerase and terminal Transferase) as a substrate.

For example, this derivative can be coupled to poly-dA or poly-A (eg, 260NT average length, Amersham Bioscience) by hybridization. Both single and multiple (dUTP-AA-PEG-biotin) 2- (dT31-biotin) 2-SA-Cy2 molecules can be coupled to a poly-dA molecule. The ratio is determined by the concentration ratios. Other oligonucleotides, for example labeled with dyes, together with (dUTP-AA-PEG-biotin) 2- (dT31-biotin) 2-SA-Cy2 can be coupled to the same strand of the poly-dA or poly-A, the ratios between individual molecules are variable. This makes it possible to produce a polyfunctional nuc macromolecule. The great advantage of such a nuc-macromolecule consists in a readily cleavable macromolecular labeling: the labeled oligonucleotides hybridized to the poly-dA or poly-A strands can be denatured be solved. By choosing the length of the labeled oligonucleotides, the Tm of these oligonucleotides can be adapted for the respective requirements of the reversible label. The rules for Tm calculation are known in the art ("Molecular Cloning", J. Sambrook, vol. 1-3, 2001). For example, dT ₂₅ oligonucleotides labeled with a Cy3 molecule can be coupled to poly-dA. Through the use of RNA, eg poly-A, for the binding of several (dUTP-AA-PEG-biotin) 2- (dT31-biotin) 2-SA molecules, the cleavage can be carried out by an RNase.

There Streptavidin 4 binding sites for Biotin has a mixture of nuc macromolecules, forming 4 binding sites are occupied differently. This mixture can with different Funds are separated. One possibility is in isolation of nuc macromolecules, which carry at least one oligo-dT31, for example by a Absorbing on an anion exchanger (e.g., a DEAE-cellulose column). On Gel electrophoresis is suitable for the separation of individual derivatives.

Also, longer nucleic acid chains carrying a biotin molecule, for example poly-dA-biotin, prepared, for example, by a terminal coupling of ddUTP-18-biotin through a TdT-dependent reaction ("Molecular Cloning", J. Sambrook, Vol. 3, 2001) can be similarly coupled to the streptavidin to form molecules having an average composition of (dUTP-AA-PEG-biotin) _N- (nucleic acid chain-biotin) _M -SA. Both single-stranded and double-stranded nucleic acid chains can be coupled. The length of the coupled nucleic acid chains may be between 10 and 100, 100 and 1000 nucleotides.

The hybridized oligonucleotides carrying a dye may be attached The poly-dA strand can also be covalently bound by cross-linkers.

Example 8.11:

(dUTP-AA-SS-PEG-biotin) 4-SA and (dUTP-AA-SS-PEG-biotin) 4-SA-Cy2, 29

The Coupling of dUTP-AA-SS-PEG-biotin to SA or to SA-Cy2 was like for (dUTP-AA-PEG-biotin) 4-SA described carried out. The starting materials used were streptavidin and dUTP-AA-SS-PEG-biotin.

It a compound was obtained which has a nucleotide functionality, a long macromolecular linker and a macromolecular marker component wearing. The linker component and the marker component can be of the nuk component are cleaved under mild conditions.

The The product of the reaction is by definition a nuc macromolecule: the linker length is significantly larger than 30 atoms, the marker component is macromolecular. It can be representative considered for nuc macromolecules which are a fissile compound in mild conditions bear the linker component.

These Compound is derived from polymerases (e.g., Klenow exo minus polymerase and terminal transferase) as a substrate.

Example 8.12: Substrate Properties of nuc macromolecules

This Example is not intended to be limiting the possible Marking reactions serve, but only differences in illustrate the substrate properties.

When Matrices served both short oligonucleotides and poly-dA. Primers and oligonucleotides were synthesized by MWG-Biotech, Germany.

Reactions were performed in 20mmol / L Tris buffer, pH 8.5, 5mmol / L MgCl ₂ , 10% glycerol. The concentrations of the primers were 1 μmol / l, those of the oligonucleotides 1 μmol / l and the concentration of the poly-dA, 0.1 μg / μl (for the concentration ratios of the solid phase see below). The polymerase used was Klenow Exo minus 1 unit / 100 μl (Amersham). The concentrations of nucleotides were 20 μmol / L for conventionally modified nucleotides and 5 μmol / L for nuc macromolecules. Unmodified nucleotides were used in concentrations of 50 μmol / l.

First, primers were hybridized to the respective template: The reaction mixture without polymerase was heated to 75 ° C and cooled to 37 ° C for 5 min. Thereafter, the polymerase was added. All Reactions were carried out for 1 h at 37 ° C. The reactions were stopped by addition of EDTA (final concentration of 10 mmol / l).

To some approaches was after stopping the reaction streptavidin, up to final concentration of 1 mg / ml, and the mixture was incubated for a further 10 min at 37 ° C. Thereby can already incorporated nucleotides carrying a biotin with streptavidin react and thus connect streptavidin and oligonucleotide. These Experiments are suitable as a control for the running properties of modified primers.

To labeled accordingly was mercaptoethanol (to 20mmol / l final concentration) was added and the respective batch was 10 min at 37 ° C incubated. Mercaptoethanol was added during some attacks during other approaches also added after the reaction.

The Analysis of the reaction was carried out by means of denaturing gel electrophoresis, 20% polyacrylamide gel, 50 mmol / l Tris-HCl, pH 8.7, as described in "Gel electrophoresis of nucleic acids ", Ed. D. Rickwood, 1990. For denaturing the samples instead of 7M urea increased gel-electrophoresis temperature used (60 ° C). Electrophoresis was carried out in Biorad gel chambers (Protean 3), 200V, about 1h. The visualization was carried out on a UV-Vis gel documentation system (Biorad).

Example 8.12A, 30

substrate properties of nuc macromolecules

• sequences:
• Primer: Primer dT ₃₅ -5'-Cy3 (dT35-Cy3): 5'Cy3-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3 '
• Die: Poly-dA (Amersham), an average of 270 nucleotides long.
• Nucleotides: (dUTP-AA-SS-PEG-biotin) 4-SA, (dUTP-16-biotin) 4-SA, dUTP Biotin-16-

Legend 30 :

Tracks 1-9:

• 1) conductors: T-7-20-Cy3, dT35-Cy3, dT40-Cy3, DT50-Cy3
• 2) (dUTP-AA-SS-PEG-biotin) 4-SA + dT35-Cy3 + poly-dA + polymerase
• 3) (dUTP-AA-SS-PEG-biotin) 4-SA + dT35-Cy3 + poly-dA
• 4) (dUTP-16-biotin) 4-SA + dT35-Cy3 + poly-dA + polymerase
• 5) (dUTP-16-biotin) 4-SA + dT35-Cy3 + poly-dA
• 6) (dUTP-16-biotin) 4-5A-Cy3 + dT35-Cy3 + poly-dA

In the control approach, traces 7-9:

• 7) dUTP-16-biotin + dT35-Cy3 + poly-dA + polymerase, Incubation 1 hr. 37 ° C afterwards + EDTA up to 10mmol / l final concentration, then + streptavidin 10 min 37 ° C
• 8) dUTP-16-biotin + dT35-Cy3 + poly-dA + polymerase, incubation 1 H. 37 ° C afterwards + EDTA up to 10mmol / l final concentration,
• 9) Conductors: T-7-20-Cy3, dT35-Cy3, dT40-Cy3, dT50-Cy3

One Nuc-macromolecule (dUTP-AA-SS-PEG-biotin) 4-SA, is incorporated into the primer (lane 2). The labeled primer has one after incorporation of a nuc macromolecule strongly changed electrophoretic mobility. Has only the presence of nuc macromolecules no influence on the primer (lane 3).

One conventionally modified nucleotide, (dUTP-16-biotin) 4-SA, with a macromolecular marker is not incorporated into the primer (Lane 4). Despite the presence of the polymerase in reaction 4 (lane 4) no differences between lane 4 and lane 5 can be detected.

track Figure 6 shows the position of the conventionally modified nucleotide with a macromolecular marker, (dUTP-16-biotin) 4-SA-Cy3, the upper band, and the position of the labeled primer (the lower Band).

Lane 7 shows the result of incorporation of dUTP-16-biotin with a subsequent reaction with the Streptavidin: the biotin-mimicked primers react with streptavidin and change their running properties. The unmodified primers retain their electrophoretic properties.

track 8 shows the result of the incorporation reaction of a conventionally modified one Nucleotides, dUTP-16-biotin. You see a common primer band, which is due to the incorporation of dUTP-16-biotin into the primer. The extension Primer is restricted, because dUTP-16-biotin are not incorporated indefinitely one after the other On average, about 3 dUTP analogs are incorporated, so that the length of Average primer rises to 38 NT. As expected, the leads Incorporation of conventionally modified nucleotides with a low molecular weight Marker does not cause a strong change in the electrophoretic mobility the primer.

In properties of the nuc macromolecules (dUTP-AA-SS-PEG-biotin) 4-SA, compared with those of conventionally modified nucleotides. It is clear that the Coupling of a macromolecular marker to a commercially acquired dUTP-16-biotin to complete Loss of substrate properties of the nucleotides leads. traces However, FIGS. 7 and 8 show that the Polymerase is well able to dUTP-16-biotin without macromolecular Insert markers into the primer. The coupling of streptavidin the biotin after the incorporation reaction leads to said changes in primer properties.

in the Contrary to this Nuc-macromolecules (dUTP-AA-SS-PEG-biotin) 4-SA in the primer without difficulty the polymerases are incorporated. The appearance of several gangs in the gel lead the Inventor of a multiple incorporation of nuc macromolecules in the Primer (3 bands) back.

Example 8.12B, 31 :

Comparison of substrate properties of nuc macromolecules, Incorporation reaction in the solution and at a fixed phase

• sequences: Primer: (dT35-Cy3) Primer dT-35-5'-Cy3: 5'Cy3-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3 '
• Die: Poly-dA (Amersham), an average of 270 nucleotides long. Oligo dA50-3'-TEG-biotin (MWG-Biotech)
• Nucleotides: (dUTP-AA-SS-PEG-biotin) 4-SA, (dU-AA-PEG-biotin) 4-SA
• Streptavidin-polystyrene Particle, 2.17μ, Spherotech Inc,

Preparation of streptavidin-polystyrene Particle (solid phase).

Three approaches were prepared in the same way.

ever 0.5 ml of the solution with beads (in manufacturer buffer) was centrifuged briefly and pellet pellet was in 100μl Incorporation buffer (20 mmol / l Tris, pH 8.5, 5 mmol / l MgCl2) resuspended. Subsequently were 100μl Oligo dA50-3'-TEG-biotin Conc. 50μmol / l added and stirred for 1 h at RT. During this time, the oligo-dA molecules bind to the beads. Subsequently were globule centrifuged briefly and washed three times in the built-in buffer. final volume the solid phase amounted to 100 μl each. This amount of oligo-dA50 solid phase can hybridize primer dT-35-Cy3 in 2 μmol / L.

The Hybridization of primer dT-35-Cy3 was carried out at 40 ° C for 10 min, with following Cooling to RT within 10 min. All other steps became the same in all approaches carried out.

Legend, 31 :

Tracks 1-10:

• 1) conductors: dT35-Cy3, dT40-Cy3,

Reactions in the liquid phase:

• 2) (dUTP-AA-PEG-biotin) 4-SA + dT35-Cy3 + poly-dA + polymerase
• 3) (dUTP-AA-PEG-biotin) 4-SA + dT35-Cy3 + poly-dA
• 4) (dUTP-AA-SS-PEG-biotin) 4-SA + dT35-Cy3 + poly-dA + polymerase, Incubation for 1 h at 37 ° C, then + EDTA
• 5) (dUTP-AA-SS-PEG-biotin) 4-SA + dT35-Cy3 + poly-dA + polymerase, Incubation for 1 h at 37 ° C, then + EDTA, then + Mercaptoethanol up to 200mmol / L (final concentration), for 30 min.
• 6) (dUTP-AA-SS-PEG-biotin) 4-SA + dT35-Cy3 + poly-dA + EDTA

Reactions at the solid phase:

• 7) (dUTP-AA-SS-PEG-biotin) 4-SA + dT35-Cy3 + Oligo dA50 solid phase + polymerase, incubation for 1 h at 37 ° C, then + EDTA
• 8) (dUTP-AA-SS-PEG-biotin) 4-SA + dT35-Cy3 + oligo-dA50-solid phase + Polymerase, incubation for 1 h at 37 ° C, then + EDTA, then + mercaptoethanol up to 200mmol / L (final concentration), for 30 min.
• 9) (dUTP-AA-SS-PEG-biotin) 4-SA + dT35-Cy3 + oligo-dA50 solid phase, before electrophoresis + EDTA
• 10) conductors: dT35-Cy3, dT40-Cy3,

you clearly sees the result of the incorporation reaction of nuc macromolecules in traces 2, 4, 5, 7, 8. Both in the solution, as well as running on the solid phase the enzymatic labeling reaction is good.

To Add mercaptoethanol to EDTA stopped reactions the cleavage of linker components with the bound streptavidin from the primers. This leads to restore the electrophoretic properties of the primers. The shift of the primer bands in lanes 5 and 8 is by multiple Incorporation of nuc macromolecules to justify in the primer. Indeed, After cleavage, the primer bands are at the level of dT40-Cy3 (see ladder). This means that until to 5 nuc-macromolecules while the reaction were incorporated into the primer.

Example 8.13:

Synthesis of dUTP-AA-SS-R-PEG-Oligo-dT31-Cy3 ( 32 )

Preparation of SH-R-PEG-Oligo-dT ₃₁ -Cy3 ( 33 ):

To 200 μl of a 100 μmol / l solution of 3'-amino-oligo-dT ₃₁ -Cy3 in 50 mmol / l borate buffer, pH 9, was added NHS-PEG-maleimide until the concentration of 20% (w / v) has been achieved. The mixture was stirred vigorously at 40 ° C for 2 h. The separation of the maleimide-PEG-oligo-dT ₃₁ -Cy3 from the excess of PEG derivative was carried out by DEAE-cellulose column chromatography: The reaction mixture was applied to the column in 10 mmol / l borate, pH 9, and with 20 column volumes of 50 mmol / l borate, pH 9, washed. Elution of maleimide-PEG-oligo-dT ₃₁ -Cy3 from the column was carried out with 1 M NaCl in 50 mmol / l borate, pH 9. The eluate was first concentrated by ultrafiltration (MWCO 3000) and the product was dissolved in 20 mmol / l Borate buffer, pH 9.0, rebuffered. The maleimide-PEG-oligo-dT ₃₁ -Cy3 was separated from oligo-dT ₃₁ -Cy3 on preparative 15% polyacrylic gel by electrophoresis, isolated from the gel and dissolved in 50 mmol / l borate buffer, pH 9.0.

To solution with maleimide-PEG-oligo-dT ₃₁ -Cy3, DTT was added to a concentration of 0.5 mol / L and the mixture was stirred at RT for 16 h. Reaction of DTT with maleimide gives rise to SH-R-PEG-oligo-dT ₃₁ -Cy3. This material was separated on DTT excess DEAE column. The reaction mixture was applied to the column in 50 mmol / l Na acetate, pH 6.0, and washed with 20 column volumes of 50 mmol / l Na acetate, pH 6.0. Elution of SH-R-PEG-oligo-dT ₃₁ -Cy 3 from the column was carried out with 1 mol / l NaCl in 50 mmol / l Na acetate, pH 6.0. The eluate was first concentrated by ultrafiltration (MWCO 3000) and the product, SH-R-PEG-oligo-dT ₃₁ -Cy3, was rebuffered in 50 mmol / l borate buffer, pH 9.0. The concentration of SH-R-PEG-oligo-dT ₃₁ -Cy3 was 100 μmol / l.

To this solution were 50 equivalents to dUTP-AA-PDTP (synthesized as in Example 7A). To Separation on DEAE-cellulose for 3 h at RT: The mixture was in 50 mmol / l Na acetate buffer, pH 6.0, applied to the column, and with 20 column volumes 50 mmol / l Na-acetate, pH 6.0, washed. Elution of dUTP-AA-PDTP carried out with 0.3 mol / l NaCl in 50 mmol / l Na-acetate, pH 6.0, elution of dUTP-AA-SS-R-PEG-oligo-dT31-Cy3 was carried out with 0.8 mol / l NaCl in 50 mmol / l Na acetate, pH 6.0. The eluate was first concentrated by ultrafiltration (MWCO 3000) and the product was rebuffered in 50 mmol / L Na acetate, pH 6.0.

The substance carries a nucleoside triphosphate functionality and a macromolecular Mar. ker functionalities (Oligo-dT31). The oligo-dT31 consists of nucleoside monophosphates, which, however, do not participate in the enzymatic reaction and have only one signal-transmitting function. To such an oligonucleotide complementary nucleic acids can be hybridized, which have a signaling function. (general rules for the hybridization of nucleic acids are known in the art, Anderson "Nucleic Acid Hybridization", 1999.).

The By definition, nucleotide is a nuc macromolecule: the linker length is significantly larger than 30 atoms, the marker component is macromolecular.

These Compound is derived from polymerases (e.g., Klenow exo minus polymerase and terminal transferase) as a substrate.

In similar Way you can Also, other oligonucleotides are coupled to the dUTP derivative. In one embodiment of the invention other homopolymer oligonucleotides, e.g. Poly-dC, poly-dG or poly-dU or poly-dA. In another embodiment, too Oligonucleotides with specific sequences can be used. By the specific sequences of oligonucleotides may be nucleic acid chains be hybridized sequence specific. For the synthesis of nuc macromolecules also oligonucleotides are used, the hairpin structures (Stemloops).

In carries this example the oligonucleotide one at the 3 'end via a Left-coupled amino group serving as a coupling site for maleimide-PEG-NHS occurs, and the Cy3 fluorescent dye. Also other coupling groups, such as SH, carboxy, aldehyde groups can be used.

The Position of the coupling group may be at one of the ends of the oligonucleotides be in the middle of the sequence. Such oligonucleotides can from the company MWG-Biotech, Germany.

When Modifications can Oligonucleotides fluorescent dyes or other reporter groups, like biotin, carry digaxygenin. Also several modifications per an oligonucleotide are possible. For example, you can FRET pairs or fluorescent dye quanine molecule pair in an oligonucleotide inserted become.

This example shows a general possibility to further modify nucleotides. Other base-modified nucleotide analogs such as 5-propargylamino-dCTP, 5-amino-propargyl-dUTP, 7-deaza-aminopropargyl-dGTP, and 7-deaza-aminopropargyl-dATP may also be modified as noted above. Both ribonucleotides, 2'-deoxyribonucleotides can be used, 18 to 21 , Other base-modified nucleotides can be used in a similar manner.

By adding poly-dA or poly-A, several dUTP-AA-SS-R-PEG-oligo-dT31-Cy3, eg 10-20, can be coupled to a nuc macromolecule. This results in a nuc macromolecule with linearly arranged nuc-linker components ( 34 ).

By Addition of other modified oligonucleotides attached to poly-dA or bind poly-A can, can also other modifications are introduced, e.g. Fluorescent label.

Literature:

• Boom, R., C.J. Sol, M.M. Salimans, C.L. Jansen, P.M. Wertheim-van Dillen, and J. van der Noordaa. 1990. Rapid and simple method for purification of nucleic acids. Clin Microbiol 28: 495-503.
• Boom, R., C. Sol, M. Beld, 7. Weel, J. Goudsmit, and P. Wertheim-van Dillen. 1999. Improved silica-guanidinium thiocyanate DNA isolation procedure based on selective binding of bovine alpha-casein to silica particles. J Clin Microbiol 37: 615-9.
• Capaldi, D.C., D.L. Cole, and V.T. Ravikumar. 2000. Highly efficient solid phase synthesis of oligonucleotide analogs containing phosphorodithioate linkages. Nucleic Acids Res 28: E40.
• Chiu, S.K., M.Hsu, W.C. Ku, C.Y. Tu, Y.T. Tseng, W.K. Lau, R. Y. Yan, J.T. Ma, and C.M. Tzeng. 2003. Synergistic effects of epoxy- and amine-silanes on microarray DNA immobilization and hybridization. Biochem J 374: 625-32.
• Damha, M.J., P.A. Giannaris and S.V. Zabarylo. 1990. An improved procedure for derivatization of controlled-pore glass beads for solid-phase oligonucleotide synthesis. Nucleic Acids Res 18: 3813-21.
• Dederich, DA, G. Okwuonu, T. Garner, A. Denn, A. Sutton, M. Escotto, A. Martindale, O. Delgado, DM Muz ny, RA Gibbs, and ML Metzker. 2002. Glass bead purification of plasmid template DNA for high throughput sequencing of mammalian genomes. Nucleic Acids Res 30: e32.
• Devivar, R.V., S.L. Koontz, W.J. Peltier, J.E. Pearson, T.A. Guillory, and J.D. Fabricant. 1999. A new solid-phase support for oligonucleotide synthesis. Bioorg Med Chem Lett 9: 1239-42.
• Diehl, F., S. Grahlmann, M. Beier, and J.D. Hoheisel. 2,001th Manufacturing DNA microarrays of high spot homogeneity and reduced background signal. Nucleic Acids Res 29: E38.
• Dolan, P.L., Y. Wu, L.K. Ista, R.L. Metzenberg, M.A. Nelson, and G.P. Lopez. 2001. Robust and efficient synthetic method for forming DNA microarrays. Nucleic Acids Res 29: E107-7.
• Habus, I., J. Xie, R.P. Iyer, W.Q. Zhou, L.X. Shen and S. Agrawal. 1998. A mild and efficient solid-support synthesis of novel oligonucleotides conjugates. Bioconjug Chem 9: 283-91.
• Hackler, L., Jr., G. Dorman, Z. Kele, L. Urge, F. Darvas, and L. G. Puskas. 2003. Development of chemically modified glass surfaces for nucleic acid, protein and small molecule microarrays. Mol Divers 7: 25-36.
• Ligler, J.J.C.F.S. 1999. Comparison of chemical cleaning methods of glass in preparation of silanization. Biosensors & Bioelectronics 14: 683-688.
• Maitra, R., and A.R. Thakur. 1994. Multiple fragment ligation on glass surface: a novel approach. Indian J Biochem Biophys 31: 97-9.
• Manko, M.A., R. Chipperfield, and H.C. Birnboim. 1982. A procedure for the largescale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder. Anal Biochem 121: 382-7.
• Merkelbach, S., J. Gehlen, S. Handt, and L. Fuzesi. 1997. Novel enzymes immunoassay and optimized DNA extraction for the detection of polymerase-chain-reaction-amplified viral DNA from paraffin-embedded tissue. At J Pathol 150: 1537-46.
• Miller, D.N., J.E. Bryant, E.L. Madsen, and W.C. Ghiorse. 1999th Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples. Appl Environ Microbiol 65: 4715-24.
• Mygind, T., L. Ostergaard, S. Birkelund, J.S. Lindholt, and G. Christiansen. 2003. Evaluation of five DNA extraction methods for purification of DNA from atherosclerotic tissue and estimation of prevalence of Chlamydia pneumoniae in a Danish population undergoing vascular repair. BMC Microbiol 3:19.
• Pon, R.T. 1993. Solid-phase supports for oligonucleotide synthesis. Methods Mol Biol 20: 465-96.
• Pon, R.T., N. Usman, and K.K. Ogilvie. 1988. Derivatization of controlled pore glass beads for solid phase oligonucleotide synthesis. Biotechniques 6: 768-75.
• Sinha, N.D. 1993. Large-scale oligonucleotide synthesis using the solid-phase approach. Methods Mol Biol 20: 437-63.
• Smith, L.S., T.L. Lewis, and S.M. Matsui. 1995. Increased yield of small DNA fragments purified by silica binding. Biotechniques 18: 970-2, 974-5.
• Song, Q., Z. Wang, and Y.S. Sanghvi. 2003. A short, novel, and cheaper procedure for oligonucleotide synthesis using automated solid phase synthesizer. Nucleosides Nucleotides Nucleic Acids 22: 629-33.
• Taylor, S., S. Smith, B. Windle, and A. Guiseppi-Elie. Of 2003. Impact of surface chemistry and blocking strategies on DNA microarrays. Nucleic Acids Res 31: e87.
• Veiko, V.P., K.I. Ratmanova, A.S. Osipov, M.T. Bulenkov, and V. V. Pugachev. 1991. [Directed introduction of amino groups by internucleotide phosphate group during solid-phase synthesis of oligodeoxyribonucleotides. Preparation of biotinylated oligonucleotide probes]. Bioorg Khim 17: 685-9.
• Vogelstein, B., and D. Gillespie. 1979. Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci U S A 76: 615-9.
• Vu, H., C. McCollum, C. Lotys, and A. Andrus. 1990. New Reactants and solid support for automated oligonucleotide synthesis. Nucleic Acids Symp Ser: 63-4.
• Zeillinger, R., C. Schneeberger, P. Speiser, and F. Kury. 1,993th A simple method for isolation of DNA from blood clots suited for use in PCR. Biotechniques 14: 202-3.

Annex 1:

modification the polymerase

Spalte A – Bezeichnung der Oligo
Spalte B – Hersteller
Spalte C – Sequenz der Oligo
Hersteller 1: MWG-Biotech, Deutschland, synthetisiert.
Hersteller 2: Thermo electron corporation GmbH, Ulm, Deutschland.
bei Oligo 31 ist die Primerbindungsstelle unterstrichen. Tabelle 19 Liste der markierten Oligonukleotiden, die im Test 1.3 eingesetzt wurden.

Spalte A – Bezeichnung der Oligo
Spalte B – Hersteller
Spalte C – Sequenz der Oligo
Hersteller 1: MWG-Biotech, Deutschland, synthetisiert.The modification of the polymerase is preferably a covalent modification. Examples of such a modification is an alkylation on the SH group, for example with alpha-halogeno-acetyl derivatives, such as, for example, iodoacetamide and its derivatives, iodoacetate and its derivatives, or with N-maleimide derivatives, further selective SH groups Reagents are described in "Chemistry of protein conjugation and crosslinking" by Shan S. Wong, 1993, CRC Press Inc. This modification can also be done by a fluorescent dye Activated fluorescent dyes that selectively react with SH groups are commercially available, eg from Molecular Probes Inc. In a preferred embodiment of the invention, a selective modification of the Klenow fragment exo-minus of the DNA polymerase to the SH group of the cysteine is carried out The cyst-modified Klenow fragment Exo-minus of the DNA polymerase may, in one embodiment instead of an unmodified Klenow fragment exo-minus the DNA polymerase can be used in the enzymatic incorporation reaction. Appendix 2 Table 18, Oligonucleotides

Column A - name of the oligo
Column B - manufacturer
Column C - sequence of oligo
Manufacturer 1: 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 - name of the oligo
Column B - manufacturer
Column C - sequence of oligo
Manufacturer 1: MWG-Biotech, Germany, synthesized.

Appendix 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:
--U ----- U ---- C ------: 180
- U - U - U - UC ------: 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
----- U - U - UC ------: 115
--U ----- U - UC ------: 162
--U - U ----- UC ------: 135
- U - U - U ---- C ------: 138
Compare to correct sequence
- U - U - U - UC ------: 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 pictures of a sequence-specific installation reaction. Shown are sections of the images of cycles 1 to 6 in Example 6.1 MFC 1330 Channel B, Pos 2.

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 , Slides, 4 , microchannel

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

4a shows schematically the components of the detection apparatus: 1 , Camera, 2 , Optical path for fluorescence signals, 3 , Color divider with blocking filter and band filter, 4 , Lens, 5 , Stage, 6 , condenser, 7 , Light path for transmitted light, 8th , 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 schematic representation of image recordings of the surface:
1 , Lens, 2 , Excitation light, 3 , Immersion oil, 4 , Cover glass (roof of the microchannel), 5 , nucleic acid chains fixed on the surface, 6 , Microchannel with a solution, 7 , Slide (bottom of the microchannel), 8th , Focus 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 detected by the coverslip surface.

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 excerpts from the surface photographs in Example 5.1.2
Panel A shows surface after testing non-specific 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
Panel A shows 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

14 Schematic representation of a reversibly labeled dUTP analogue s. Example 7E

Claims (112)

A method for parallel sequence analysis of nucleic acid sequences (Nucleic acid chains, NSKs) by optical means, which includes the following steps: 1. Providing a solid phase 2. Binding of to be analyzed nucleic acid chains to the solid phase to form extensible primer-template complexes, Third execution a cyclic reaction with those fixed on the solid phase Primer-template complexes, including the following steps: 3.1 enzymatic incorporation of labeled nucleotides on the formed Primer-template complexes 3.2 Washing 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 the solid phase 3.6 if necessary repeat the steps 3.1 to 3.5 4. Reconstruction of the sequences of single nucleic acid chains from the signals obtained in step 3.3 Fixed phase for Method for parallel analysis of a plurality of individual nucleic acid molecules with optical Composition according to claim 1, wherein the surface of the solid phase has a property the low non-specific binding of labeled components. The solid phase of claim 2, wherein the non-specific binding of labeled components is a Bin 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 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 A method of producing the solid phase according to claims 1 to 16, wherein the preparation is as follows Steps includes: 1. Removal of the outer layer of the solid phase 2. Coupling of nucleic acid chains to 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. 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 a Length between Have 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. The fixed phase with fixed nucleic acid chains according to claims 1 to 56, wherein the solid phase is for electromagnetic radiation having 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 Composition according to claim 1, wherein the solid phase according to claims 2 to 59 is used. 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 multiplicity of individual nucleic acid molecules by optical means according to claims 60 to 85, wherein 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. 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. A method for the parallel analysis of a plurality of individual nucleic acid molecules with optical means according to claims 82 to 101, wherein the number of repetitions of steps 3.1 to 3.5 is between 2 and 10 lies. 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 Means according to claim 1, 60 to 105, wherein as labeled nucleotides Conventionally modified nucleotides are used. Method for the parallel analysis of a multitude single nucleic acid molecules with optical Means according to claim 1, 60 to 105, wherein as labeled nucleotides Nuc-macromolecules be used. 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. Method for analyzing nucleic acid chains with the solid phase according to claims 2 to 59, wherein the nucleic acid chains to be analyzed on the solid phase are fixed and analyzed by hybridization of complementary labeled nucleic acid chains he follows. Method for analyzing nucleic acid chains with the solid phase according to claims 2 to 59, wherein on the solid phase nucleic acid strands in a predefined Arrangement are fixed and the nucleic acid chains to be analyzed analyzed by hybridization to the fixed nucleic acid strands become.