DE10120797B4 - Method for analyzing nucleic acid chains - Google Patents

Abstract

A method for the highly parallel sequence analysis of nucleic acid sequences or nucleic acid chains (NSKs), in which
Fragments (NSKFs) of single-stranded NSKs of about 50 to 1000 nucleotides in length, representing overlapping partial sequences of the total sequences;
the NSKFs binds to a single primer or several different primers in the form of NSKF primer hybridization products on a reaction surface in a random array;
Perform a cyclic assembly reaction of the complementary strand of NSKFs using one or more primers and one or more polymerases by
a) to the immobilized NSKFs a solution containing one or more polymerases and one to four modified nucleotides (NTs *), which are labeled with fluorescent dyes, wherein the simultaneous use of at least two NTs * each on the NTs * fluorescent dyes are chosen so that the NTs * used can be distinguished from one another by measuring different fluorescence signals, the NTs * being structurally modified at the base in such a way that the NTs * are structurally modified.

Description

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. On this surface Many single nucleic acid molecules are immobilized. All these nucleic acid molecules are exposed to the same conditions, so that at all nucleic acid molecules simultaneously a build-up reaction can take place.

• 1) Immobilisierung der Nukleinsäureketten auf einer planen Oberfläche.
• 2) Durchführen einer zyklischen Aufbaureaktion, wobei jeder Zyklus aus folgenden Schritten besteht: a) Zugabe einer Lösung mit markierten Nukleotiden (NTs*) und Polymerase zu immobilisierten Nukleinsäureketten, b) Inkubation der immobilisierten Nukleinsäureketten 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. Gegebenenfalls erfolgt eine mehrfache Wiederholung des Zyklus.
• 3) Analyse der detektierten Signale der einzelnen Moleküle.
• 4) Rekonstruktion der Sequenzen aus den Einzeldaten.

The method essentially comprises the following steps:

• 1) immobilization of the nucleic acid chains on a plane surface.
• 2) carrying out a cyclic synthesis reaction, each cycle consisting of the following steps: a) addition of a solution with labeled nucleotides (NTs *) and polymerase to immobilized nucleic acid chains, b) incubation of the immobilized nucleic acid chains with this solution under conditions sufficient to extend the complementary nucleic acid chains C) washing, d) detection of signals from single molecules, e) removal of the label from the incorporated nucleotides, f) washing. If necessary, there is a multiple repetition of the cycle.
• 3) Analysis of the detected signals of the individual molecules.
• 4) Reconstruction of the sequences from the individual data.

1. Abbreviations and term explanations

• DNA

  - Deoxyribonucleic acid different Origin and different length: (genomic DNA, cDNA, ssDNA, dsDNA)

  RNA

  - Ribonucleic acid (usually mRNA)

  polymerases

  - Enzymes that are complementary nucleotides into a growing DNA or RNA strand (e.g. DNA polymerases, reverse transcriptases, RNA polymerases)

  dNTP

  2'-deoxynucleoside triphosphates for DNA polymerases and reverse transcriptases

  NTP

  - Nucleoside triphosphates for RNA polymerases

  NT

  - natural nucleotide, mostly dNTP, if not express otherwise marked.

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 nucleotide monophosphates.

NT*

– modifiziertes Nukleotid, meist dNTP, wenn nicht ausdrücklich anders gekennzeichnet. NTs* bedeutet: modifizierte Nukleotide

NSK

– Nukleinsäurekette. DNA oder RNA in ihrer ursprünglichen Länge

NSKF

– Nukleinsäurekettenfragment, NSKFs – Nukleinsäurekettenfragmente. Fragmente von DNA oder RNA, die nach einem Fragmentierungsschritt entstehen.

In the text, the majority of abbreviations are formed by using the suffix "s", "NT" stands for "nucleotide", for example, "NTs" stands for several nucleotides.

NT *

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

NSK

- Nucleic acid chain. DNA or RNA in their original length

NACF

- Nucleic acid chain fragment, NSKFs - nucleic acid chain fragments. Fragments of DNA or RNA arising after a fragmentation step.

Plans Surface: Surface, which preferably has the following features: 1) It allows several individual molecules preferably more than 100, more preferably more than 1000, with the respective given lens surface distance to detect simultaneously at a lens position. 2) The immobilized single molecules are in the same focal plane, which are reproducible can be.

steric Obstacle: Sterically demanding group, by their chemical Structure the properties of the NTs coupled to this group * so changed, that these by a polymerase in an extension reaction not sequentially can be installed.

definition Termination: Termination in this application is reversible Stop the installation of modified uncleaved NTs * designated.

This term is from the common use of the word "termination" by dideoxy-NTP in one separate conventional sequencing.

The Termination comes about after the installation of a modified NT *. The modified built-in NT * reverses one to the base coupled steric group, which inhibit the incorporation of a next complementary NT * leads to the growing strand through a polymerase.

Gene Products - At the Gene products are the primary gene products of the genes. in the Essentially, these are RNA transcripts of the mentioned Genes which also act as target sequences (or target nucleic acid sequences) be designated. These target sequences also include mRNA derived therefrom single and double-stranded cDNA, cDNA-derived RNA or cDNA amplified DNA.

SNP - single nucleotide polymorphism

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), the on based on the incorporation of chain terminators, and the Maxam-Gilbert method, the base-specific modification and cleavage of nucleic acid chains (M.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. Chr. 88 p. 10089, R.S. Drmanac et al. Science 1993 v. Chr. 260 p. 1649). This process also has problems: it can only one sequence can be determined in one approach, secondary structures to disturb the hybridization and repeats prevent the correct one Analysis.

A different possibility for sequencing, groups of (Dower US Patent 5,547,839, Canard et al. US Patent 5,798,210, Rasolonjatovo Nucleosides & Nucleotides 1999, v. 18 p. 1021, Metzker et al. NAR 1994, v. 22, p. 4259, Welch et al. Nucleosides & nucleotides 1999, v. 18, p. 197). This method is abbreviated as BASS (Base Addition Sequencing Scheme). There will be a size Number of equal single-stranded ones DNA pieces fixed on a surface at a defined location and the signal from analyzed the entirety of these many identical DNA pieces. To this fixed DNA becomes a solution Polymerase and nucleotides added so that a complementary strand can be synthesized. The polymerase should be gradual work: in each step only a single nucleotide is incorporated. This is detected, whereupon the polymerase in a next cycle the next Incorporating nucleotide.

The Method has the following disadvantages: When building the complementary strands occurs very fast desynchronization of the synthesis, so that at each Step the errors accumulate. Therefore, only very short fragments be sequenced. It should be emphasized that all the BASS methods described not based on the detection of single molecules. The signal is instead of a big one Number of identical molecules registered at a defined place of immobilized molecules. The usual in these methods Use of the terms "individual Molecules "and" Molecules "are not targeted to individual, separate molecules, but to a population, those from many identical molecules consists. Identical means in In this case, that the molecules have the same sequence.

The object of the present invention is therefore to provide a method for sequence analysis of nucleic acid chains, which does not have the disadvantages of the above-mentioned methods and above all allows a cheaper, faster and more efficient analysis of nucleic acid sequences. In particular, the method should be able to determine many sequences in parallel, preferably it should be the analysis allow very long nucleic acid chains (several Mb) in one approach.

3. Short description

• a) zu den immobilisierten NSKFs 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 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 NSKFs 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 und man aus den ermittelten überlappenden Teilsequenzen die Gesamtsequenz der NSKs bestimmt.The object is achieved by a method for sequence analysis of nucleic acid sequences (Nulcleinsäureketten, NSKs), in which one generates fragments (NSKFs) single-stranded NSKs with a length of about 50 to 1000 nucleotides, which are overlapping partial sequences of the total sequences, one the NSKFs on a Reaction surface at a density of 10 to 100 NSKFs per 100 microns ² immobilized (stationary phase), one carries out a cyclic synthesis reaction of the complementary strand of NSKFs using one or more primers and one or more polymerases by

• a) to the immobilized NSKFs a solution containing one or more polymerases and one to four modified nucleotides (NTs *), which are labeled with fluorescent dyes, wherein the simultaneous use of at least two NTs * each on the NTs * fluorescent dyes are chosen so that the NTs used can * differ from each other by measuring different fluorescence signals, wherein the NTs * are structurally modified so that the polymerase after incorporation of such NT * in a growing complementary strand is not able to another NT * incorporate into the same strand, wherein the fluorescent dye is cleavable and the structural modification is a cleavable sterically demanding ligand, one
• b) the stationary phase obtained in step a) is incubated under conditions suitable for extending the complementary strands, the complementary strands being extended by one NT * each;
• c) washing the stationary phase obtained in stage b) under conditions which are suitable for the removal of non-complementary NTs *
• d) the individual NTs * incorporated in complementary strands are detected by measuring the signal characteristic of the respective fluorescent dye, at the same time determining the relative position of the individual fluorescence signals on the reaction surface;
• e) for the production of unlabeled (NTs or) NSKFs, the fluorescent dyes and the sterically demanding ligands of the NTs attached to the complementary strand * splits, one
• f) washing the stationary phase obtained in step e) under conditions 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 NSKFs 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 and the overall sequence of the NSKs is determined from the determined overlapping partial sequences.

The preserved population of overlapping Partial sequences can be 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. Chr. 33 p. 21, Bonfield et al. NAR 1995 v. 23 p. 4992, Miller et al. J. Comput. Biol. 1994 v. Chr. 1 P. 257).

• 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 present invention also relates to the in the 7e . 7f and 7g represented nucleotides and the corresponding labeled nucleotides, which have, for example, attached to the terminal amino function fluorescent dyes, or in the 7h . 7i or 7y shown labeled nucleotides.

object The invention further provides a kit for carrying out the method Reaction surface, to carry out the reaction required, one or more polymerases, and Contains nucleotides (NTs), of which one to four are labeled with fluorescent dyes, the NTs being structurally modified (NT * or NTs *), that the Polymerase after incorporation of such NT * into a growing complementary strand unable to build another NT * in the same strand, wherein the fluorescent dye is cleavable and the structural Modification is a cleavable sterically demanding ligand. The nucleotides are preferably those mentioned above nucleotides according to the invention.

According to one particular embodiment of the invention the kit also required to produce single strands of double strands Reagents, single-stranded Nucleic acid molecules that introduced as PBS into the KSKFs be, oligonucleotide primer, to cleave the fluorescent dyes and sterically demanding Ligands required reagents and / or wash solutions.

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

The Preparation of the material to be analyzed (single- and double-stranded nucleic acid sequences) depends on the task and has the goal of a long nucleic acid chain a population of relatively small, single-stranded nucleic acid chain fragments (NSKFs) to form these fragments with one for the start of the sequencing reaction appropriate primer 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 to these molecules can expire. In principle, different types of immobilization possible, those of the objective, the nature of the NSK and the reaction depend on the polymerase used. The NSKFs are randomly distributed on the surface during immobilization, i.e. it does not have to be pay attention to an exact positioning of the individual chains. The nucleic acid chains have to go with it be fixed in such a density on the surface that a unique Assignment of the later detected signals to individual NSKFs is guaranteed.

To The preparation of the NSKFs starts with all surfaces immobilized on the surface Molecules that Sequencing reaction. The basis of sequencing is the Synthesis of the complementary strand to every single immobilized NSKF. It will be in the new synthesized strand labeled NTs * incorporated. The polymerase builds only a single tagged NT * into the growing chain. This is due to the reversible coupling one for termination leading, sterically demanding group to the NTs * achieved. The installation another marked NT * is thereby made impossible. This steric Sophisticated group is preferably a fluorescent dye.

• a) Zugabe einer Lösung mit markierten Nukleotiden (NTs*) und Polymerase zu immobilisierten Nukleinsäureketten,
• b) Inkubation der immobilisierten Nukleinsäureketten 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 immobilized nucleic acid chains,
• b) incubating the immobilized nucleic acid chains with this solution under conditions suitable for extending 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 to more than 50% of those involved in the sequencing reaction NSKFs can install a marked NT * in one cycle, preferably more than 90%.

The Number of performed Cycles depends thereby from the respective task off, is theoretically not limited and is preferably between 20 and 5000.

Then, for each fixed NSKF, its specific sequence is determined from the order of the built-in NTs *. From the overlapping NSKF sequences, the original NSK sequence can be reconstructed ("Automated DNA sequencing and analysis" page 231 et seq., 1994, M. Adams et al., Academic Press, Huang et al., Genom Res., 1999, v. 9 p. 868, Huang Genomics 1996 v. 33 p.21, Bonfield et al NAR 1995 v. 23 pp. 4992, Miller et al., J. Comput. Biol. 1994 v. 1 pp. 257). In the whole population of NSKF sequences one looks for matches / overlaps in the sequences of NSKFs. By these matches / overlaps one can bring the NSKF in a row, eg:

In The practice has involved sequencing unknown sequences proven, a length the sequenced pieces of more than 300 bp. This allows sequencing of genomes from eukaryotes in the shot-shot method.

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

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

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

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

Short description the figures

1 General Principle of Sequencing a Long Nucleic Acid Chain (Schematic)

• 1) starting material - the long to be analyzed Nucleic acid sequence overall sequence
• 2) Fragments of 50-1000 bp - the NSKFs generated from the overall sequence in the fragmentation step
• 3) Fragments with one primer each - NSKF primer hybridization products
• 4) Immobilized fragments - bound to the planar surface NSKF primer hybridization products, in this embodiment the bond is at the 3'-end the NSKFs
• 5) Add a solution with polymerases and NT * s as a first step in a cycle of sequencing reaction
• 6) Washing step - after the installation step, the surface is washed
• 7) Detection - the Signals from individual built-in NT * s are detected
• 8) Removal of the mark and that leading to the termination Group - to Continuation of the sequencing reaction, the label and removed the steric barrier

2 : Examples of the General Structure of Immolubilized Nucleic Acid Fragments

These Figure shows one to the 3 'end the NSKFs (3) coupled single primer binding site (PBS; 2) and a single primer bound to this PBS (1).

3 : Oligonucleotide construction with a primer binding site and blocked ends

One double Oligonucleotide Complex (3a) Having a Primer Binding Site (PBS) comprises can be modified on one side of the oligonucleotide on both strands (3b), so that only one PBS is ligated to the NSKF. After ligation to the double-stranded ones NSKFs and after denaturation (3c) result in single-stranded NSKFs with uniform PBS and modified ends (3d).

4 : Nucleotide tailing

To the 3 'end of a single stranded DNA fragment (NSKFs) are indicated by so-called "Tai With the aid of a terminal deoxynucleotidyltransferase, several nucleoside monophosphates are attached (guanosine monophosphates and the so-called (G) n tailing are shown.) Using uniform nucleoside monophosphates, a uniform PBS is formed.

5 : Examples of the binding of NSKFs to a gel-like reaction surface

• a) A gel (2) adheres to a solid surface (1), e.g. a polyacrylamide gel. At the continuous surface bind over on the Gel bound groups (3) (e.g., streptavidin or avidin) NSKFs (4) with a functional group suitable for immobilization (3), e.g. Biotin.
• b) arrangement as in 5b However, the binding of NSKFs occurs via a discontinuous surface composed of a number of gel beads (5), eg agarose beads, to the solid surface.

6 : Example of a flow system

A gelatinous reaction surface (1) is on a for the excitation and fluorescent light permeable solid support (2) attached. It together forms the lid of the reaction chamber (e.g. Flow Cell). The liquids in the reaction chamber can be replaced in a controlled manner, the reaction chamber together with the reservoir (3), the pump (4) and the valve (5) a flow system form. The signals of those bound in the on the reaction surface NSKF primer hybridization products (not shown here) incorporated NT * s will be over the belonging to the detection apparatus Lens of the microscope (6) detected.

7 General Structures of NT * (Labeled 2'-Deoxynucleoside Triphosphates)

• a) Schematic representation of the NT structure semitterminators, where at the base via a cleavable linker (A-E) a steric group (D) and the fluorescent marker (F) bound are. A, C, E connectors in the linker; A - linker residue after the split; B - fissile Compound / group; D - sterically demanding group leading to termination; F - fluorescent dye (Marker).
• b) Schematic representation of the NT structure in which the Fluorescent dye simultaneously the function of sterically demanding Group takes over. A, C - linker; A - linker residue after the split; B - fissile Compound / group; D - sterically demanding group leading to termination; F - fluorescent dye (Marker).
• c) Schematic representation of the structure of built-in NT * s after the cleavage step. Shown are two NT * s with the remaining linker residue (A).
• d) Schematic representation of the NT structure in which the fissile group, which at the same time the sterically demanding, leading to termination Group is, represents a part of the linker. This steric group may be a photolabile group. A, C - linker; A - linker residue after the split; B - fissile Compound / group; D - sterically demanding group leading to termination; F - fluorescent dye (Marker).
• e) Representation of preferred NT structures in which the Linker is coupled to the 5-position in the pyrimidine ring (e-1: uracil; e-2: cytosine).
• f) Representation of further preferred NT structures in which the linker is coupled to the 5-position in the pyrimidine ring (f-1 and f-2 each uracil; f-3 and f-4 each cytosine).
• g) Representation of preferred NT structures in which the Linker is coupled to the 7-position in the purine ring (g-1: adenine; g-2: guanine).
• h-j) Examples of the coupling of dyes to the linker attached to the base of the Nucleotide is coupled. (7h) dNTP-SS-TRITC; 7i) dNTP-SS-Cy3; 7y) dNTP-SS-TRITC)

8th Example of a detection device

shown is a wide-field optical detection device with a light source for exciting the fluorescence (1), a light-conducting part (2), a scanning table (3), a device for selecting the spectra (4), a detection device (5), a computer with control and analysis function (6) and a surface with immobilized NSFKs (7), which is scanned after installation of marked NT * s, where detects the fluorescence signals from individual dye molecules coupled to the NTs become.

• a) Schematic representation of a section the reaction surface (gray), which is scanned step by step. The circles correspond each recording a 2D image and represent the areas of where the fluorescence signals are detected.
• b) Representation of a photograph (a 2D image) with fluorescence signals the single, built-in NT * s. There are several signals per recording (For example, 100 to 10,000) of single molecules simultaneously be registered.
• c) detail from 8b , The section shows signals from built-in NT * s. Each of the identified signals are assigned the corresponding X, Y coordinates. Each signal has characteristic properties and can be identified by it (preferably by means of a computer program).

9 : Sequencing reaction on two separate surfaces

Of the Throughput is achieved by using two separate flow systems (e.g., flow cells (microfluidic channels, MFK)) elevated. While on the one surface Biochemical and chemical reactions take place on the other the detection performed. Subsequently swap the surfaces their positions in the flow cells.

4. Detailed description

The Abbr. "M", "mM" and "μM" stand for the units mol / l, mmol / l or μmol / l.

4.1 General principles the reaction

In the following, the general principles of the reaction are to be exemplified by the sequencing of a several Mb long piece of DNA ( 1 ). The sequencing and reconstruction of nucleic acid sequences is based on the shotgun principle ("Automated DNA sequencing and analysis" p. 231 et seq., 1994. M. Adams et al., Academic Press, Huang et al., Genome Res. 1999, 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). The sequence of a long piece of DNA is determined by the sequencing of small DNA fragments and subsequent reconstruction. The material (1) to be analyzed is prepared for the sequencing reaction by breaking it up into fragments of preferably 50 to 1000 bp in length (2). Each fragment is then provided with a primer binding site and a primer (3). This mixture of different DNA fragments is now fixed on a flat surface (4). The unbound DNA fragments are removed by a washing step. Thereafter, the sequencing reaction on the entire

Reaction surface performed. These Reaction proceeds cyclically. In the 1st step of the cycle, one becomes with a fluorescent dye marked NT * incorporated into the growing strand: This is the Reaction controlled so that in each cycle only one labeled NT * of a polymerase can be installed in the growing strand. That is through the Use of NTs * achieved a reversibly coupled, for Termination leading Bear group. The installation of another marked NT * is thereby impossible made. The polymerase and the labeled NTs * become simultaneously into the reaction (5). Thereafter, the reaction mixture removed and the surface washed in a suitable manner (6). Now follows a detection step (7): The surface will be with a for the single molecule detection suitable device (consisting of light source, microscope, camera, Scan table, computer with control and image recognition or image processing software) scanned and the signals of each, built-in marked NTs * identified. After the detection step, the mark and the leading to the termination Group removed from all built-in NTs * (8). After one subsequent Washing step can start a new cycle. For reconstruction a larger original DNA sequence (e.g., several Mb long piece of DNA) the DNA fragments should be a few hundred NT long, if one performs the reconstruction according to the shotgun principle ("automated DNA sequencing and analysis "p. 231 ff. 1994 M. Adams et al. Academic Press, Huang et al. genome Res. 1999 v. Chr. 9 p. 868, Huang Genomics 1996 v. Chr. 33 p. 21, Bonfield et al. NAR 1995 v. 23 p. 4992, Miller et al. J. Comput. Biol. 1994 v. 1 p. 257). Since only one marked NT * is installed per cycle At least 300 cycles are required for sequencing.

4.2 Selection of the material

With the aid of the method according to the invention, it is possible to use both preselected DNA sequences (eg in YAC, PAC or BAC vectors (Anand, R., et al NAR 1989, 17 pp 3425, H. Shizuya et al., PNAS 1992 v. 89 p. 8794, "Construction of bacterial artificial chromosome libraries using the modified PAC system" in "Cur rented Protocols in Human Genetics "1996 John Wiley & Sons Inc.) as well as non-preselected DNA (eg, genomic DNA, cDNA mixtures) to analyze by pre-selection, in advance relevant information, such as Sequence sections from a genome or populations of gene products, to filter out of the large amount of genetic information and thus to limit the amount of sequences to be analyzed.

4.3 Preparation of the material

The aim of the material preparation is to obtain immobilized single-stranded NSKFs with a length of preferably 50-1000 NTs, a single primer binding site and a hybridized primer. These fragments preferably have the in 2 illustrated structure. In particular, very variable constructions can be derived from this general structure. To improve the clarity, some examples now follow, the methods given may be used individually or in combination.

4.3.1 Generation short Nucleic acid chain fragments (50-1000 NTs)

Important is that the Fragmentation of the NSKs is done so that fragments are obtained the overlapping Represent partial sequences of the overall sequences. This is done by procedures reached, in which fragments of different lengths as fission products in random distribution arise.

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

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

4.3.2 Introduction of a Primary binding site in the NSKF.

The Primer binding site is a sequence section that is a selective To allow binding of the primer to the NSKF.

Out establish The simplification of the analysis is favorable, if possible uniform primer binding site is present in all NSKFs. 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.

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 a primer binding site are the ligation and nucleotide tailing to DNA fragments described.

a) Ligation:

A double-stranded oligonucleotide complex with a primer binding site is used ( 3a ). This is ligated to the DNA fragments with commercially available ligases (Molecular cloning 1989 J. Sambrook et al., Cold Spring Harbor Laboratory Press). It is important that only a single primer binding site be ligated to the DNA fragment. This can be achieved, for example, by modifying one side of the page Oligonucleotide complex on both strands ( 3b ). The results after ligation or subsequent denaturation are in 3c and 3d shown. The modifying groups on the oligonucleotide complex can serve for immobilization. The synthesis and the modification of such an oligonucleotide complex can be carried out according to standardized instructions. For example, the DNA synthesizer 380 A Applied Biosystems can be used for the synthesis. However, oligonucleotides with a specific composition, with or without modifications, are also commercially available as an order synthesis, for example from MWG-Biotech GmbH, Germany.

b) nucleotide tailing:

Instead of ligation with an oligonucleotide, several (eg between 10 and 20) nucleoside monophosphates can be linked to a terminal deoxynucleotidyltransferase at the 3 'end of an ssDNA fragment ("Molecular cloning" 1989, J. Sambrook et al., Cold Spring Harbor Laboratory Press, "Method in Enzymology" 1999 v. 303, pp. 37-38) ( 4 ), eg several guanosine monophosphates (called (G) n-tailing). The resulting fragment is used to bind the primer, in this example a (C) n primer.

4.3.3 Single-strand 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).

4.3.4 Primer for the sequencing reaction

This has the function to start at a single point of the NSKF enable. Preferably, it binds to the primer binding site in the NSKF. The Composition and the length of the primer are not restricted. Except the start function, the primer can also perform 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.

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, preferably such a functional 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 may be pre-hybridized to the fragments to be analyzed on the surface fixed with different techniques or synthesized directly on the surface (McGall et al., U.S. Patent 5,411,087, Barrett et al., U.S. Patent 5482867, Mirzabekov et al. US Patent 5981734, "Microarray biochip technology" 2000 M. Schena Eaton Publishing, "DNA Microarrays "1999 M. Schena Oxfoxd University Press, Fodor et al. Science 1991 v. Chr. 285, p. 767, Timofeev et al. Nucleic Acid Research (NAR) 1996, v. 24 p. 3142, Ghosh et al. NAR 1987 v. Chr. 15 pp. 5353, Gingeras et al. NAR 1987 v. Chr. 15 pp. 5373, Maskos et al. NAR 1992 v. 20 p. 1679).

Of the Primer or primer mix is used with NSKFs under hybridization conditions which selectively delivers it to the primer binding site of the NSKF let bind. This primer hybridization may be before, during or after immobilization of the NSKFs. The optimization of Hybridization conditions depends on the exact structure of the primer binding site and the primer and lets himself go according to Rychlik et al. NAR 1990 v. Calculate 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 introduced by ligation will, can Primers with a uniform structure can be used. The primer binding site can at her 3'-end carry a functional group, e.g. serves for immobilization. Preferably, this group is a biotin group. The primer has a complementary to the primer binding site structure.

One Example of a primer binding site and a primer is below shown.

4.3.5 Immobilization

aim immobilization is to place NSKFs on a suitable planar surface in one Way to fix that one cyclic enzymatic sequencing reaction can proceed. Surface and reaction surface are to be understood as equivalent terms in this application, except if explicit reference is made to another meaning. Serves as a reaction surface the surface a solid phase of any material. This material is preferably enzymatic reactions to inert and causes no disorders the detection. Silicone, glass, ceramic, plastic (e.g., polycarbonates or polystyrenes) or any other material containing this functional Requirements are sufficient can be used. Preferably, the surface is not deformable, because otherwise with a distortion of the signals at to reckon with the repeated detection.

If a gel-like solid phase is used, this gel may be, for example, an agarose or polyacrylamide gel. The gel is preferably freely passable for molecules of molecular mass below 5000 Da (for example, a 1 to 2% agarose gel or 10 to 15% polyacrylamide gel may be used). Such a gel surface has the advantage over other solid surfaces that the labeled NSKFs are retained on the surface as opposed to free NTs *. The immobilization of the NSKFs on the surface makes it possible to detect the fluorescence signals of incorporated NTs *. The signals of free NTs * are not detected because they do not bind to the material of the gel and thus are not immobilized. The gel is preferably attached to a solid support ( 5a ). The thickness of the gel is preferably not more than 0.1 mm. However, the money must be greater than the simple depth of field of the lens, so nonspecifically bound to the solid surface NTs * do not reach the focal plane and thus detected. If the depth of focus is, for example, 0.3 μm, the thickness of the coin is preferably between 1 μm and 100 μm. The surface can be produced as a continuous surface or as a discontinuous surface composed of individual small components (eg agarose beads) ( 5b ). The reaction surface must be large enough to be able to immobilize the necessary number of NSKFs with appropriate density. The reaction surface should preferably be no larger than 20 cm ² .

The various cycle steps require an exchange of the different reaction solutions above the surface. The reaction surface is preferably part of a reaction vessel. The reaction vessel is again preferably part of a reaction apparatus with a flow device. The flow device allows an exchange of the solutions in the reaction vessel. The replacement can be done with a computer-controlled pumping device or manually. It is important that the surface does not dry out. The volume of the reaction vessel is preferably less than 50 μl. Ideally, its volume is less than 1 μl. An example of such a flow system is in 6 given.

The Immobilization of the NSKFs is preferably carried out on one of the two Chain ends. This can be done by appropriate covalent, affine or other bindings are achieved. There are many examples of immobilization of nucleic acids (McGall et al., US Patent 5412087, Nikiforov et al., US Patent 5610287, US Pat. Barrett et al. U.S. Patent 5,482,867, 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. Chr. 73 P. 2064, Trabesinger et al. Analytical Chemistry 1999, v. 71, p. 279, Osborne et al. Analytical Chemistry 2000, v. 72, p. 3678, Timofeev et al. Nucleic Acid Research (NAR) 1996, v. 24 p. 3142, Ghosh et al. NAR 1987 v. Chr. 15 pp. 5353, Gingeras et al. NAR 1987 v. Chr. 15 p. 5373, Maskos et al. NAR 1992 v. 20 p.1679).

The NSKFs are preferably immobilized on the surface at a density of between 10 and 100 NSKFs per 100 μm ² . By way of example, two methods of immobilization are described in more detail below: In a preferred embodiment, the immobilization of the NSKFs takes place via biotin-avidin or biotin-streptavidin binding. This avidin or streptavidin is covalently bound on the surface, the 5 'end of the primer contains biotin. After hybridization of the labeled primers with the NSKFs, they are fixed on the avidin / streptavidin-modified surface. The concentration of biotin-labeled hybridization products and the time of incubation of this solution with the surface are chosen to achieve a density suitable for sequencing.

In another preferred embodiment, the primers suitable for the sequencing reaction are fixed on the surface by suitable methods before the sequencing reaction (see above). The single-stranded NSKFs with one primer binding site per NSKF are thus incubated under hybridization conditions. They bind to the fixed primer and are thereby immobilized. The concentration of the single-stranded NSKFs is chosen so as to achieve a suitable sequencing tion immobilization density of 10 to 100 NSKFs per 100 microns ² . After immobilization, unbound NSKFs are removed by a wash step. If the surface of a solid phase (eg silicone or glass) is used for immobilization, preferably a blocking solution is applied to the surface prior to step (a) in each cycle to avoid nonspecific adsorption of NTs * to the surface. These conditions for a blocking solution, for example, meets an albumin solution (BSA) with a pH between 8 and 10.

4.4 Choice of polymerase

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

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

4.5 Chemistry

4.5.1 General Principle

• 1) einzelne Zugabe von NT, so daß die Polymerase nur einen einzigen Schritt macht und stoppt (Substrat Limitierung),
• 2) Änderungen der äußeren Bedingungen (Temperatur, pH, Salz usw.), so daß die Polymerasen ihre Funktion anders ausüben (s. Optimierung der Reaktionsbedingngen),
• 3) durch bestimmte kompetetive und nichtkompetetive Inhibitoren (als Beispiel seien antivirale Medikamente genannt)
• 4) Durch bestimmte Mutationen und andere Veränderungen an der Polymerase als solcher,
• 5) Durch die Struktur der NT. Diese Strukturänderungen können sein: a) irreversibel (wie z.B. bei dideoxyNTPs) b) reversibel.

It is important for the sequencing reaction that each incorporated NT * be identified. A prerequisite for this is that only a single NT * per cycle is incorporated into the nucleic acid chain. If a polymerase incorporates several NTs * in succession in the same cycle, this leads to an error in the sequence determination. For this reason, one must control the installation of NTs *. In this context, various ways of controlling the enzymatic reaction are conceivable, such as

• 1) single addition of NT, so that the polymerase only makes a single step and stops (substrate limitation),
• 2) changes in the external conditions (temperature, pH, salt, etc.), so that the polymerases perform their function differently (see Optimization of Reaktionsbedingngen),
• 3) by certain competitive and non-competitive inhibitors (for example antiviral drugs)
• 4) By certain mutations and other changes to the polymerase as such,
• 5) Through the structure of the NT. These structural changes may be: a) irreversible (such as in dideoxyNTPs) b) reversible.

Preferably the control is carried out by a specific structure of NTs *, where reversible structural changes are particularly preferred. The reversible structural changes For example, by attaching a sterically demanding Ligands, i. a ligand, the polymerase from the incorporation of a another NT stops. The control as a result of steric blocking of the enzymatic Reaction by reversible semiterminators is described in more detail below. According to one preferred embodiment The invention relates to the bases by coupling a fluorescent dye structurally changed or modified.

There are several principal variants of the NT * structure which hinder or stop the extension reaction: For example, reversible 3'-OH modified NTs have been described in the BASS method (Dower US Patent 5,547,839, Canard et al., US Pat Patent 5,798,210, Rasolonjatovo Nucleosides & Nucleotides 1999, v. Chem. 18 p. 1021, Metzker et al. NAR 1994, v. 22, p. 4259, Welch et al. Nucleosides & Nucleotides 1999, v. 18, p. 197). The cleavage is said to be photochemical under mild conditions (Dower US Patent 5,547,839, Welch et al., Nucleosides & Nucleotides 1999, v. 18, p197) or chemically (Canard et al., US Patent 5,798,210, Rasolonjatovo Nucleosides & Nucleotides 1999, v. 18 p.1021).

The Synthesis of 3'-OH-modified photochemically cleavable NTs * is very expensive. The polymerases have a very different affinity for these nucleotide analogs on, so that the nucleic acid synthesis very uneven or at some DNA sites does not expire at all (Metzker et al NAR 1994 v. 22 p 4259, Welch et al Nucleosides & Nucleotides 1999, v. 18 p. 197). For this reason, these analogs are only partially suitable for the Sequencing reaction. A cleavable 3'-ester linkage (Canard et al., US Patent 5,798,210) comes as a basis for a reversible termination of the synthesis also not considered. Most polymerases cleave upon availability of a next complementary NT 3'-OH ester compounds, So that the bonded to the 3'-OH group Mark in the solution is released and can no longer act as a terminator (Rasolonjatovo et al. Nucleosides & nucleotides 1999, v. 18 p. 1021, Canard et al. PNAS 1995 v. 92 p. 10859). In Positions where a polymerase several equal NTs * consecutively can install leads that to a faulty signal.

According to the invention for the Sequencing NTs * with a sterically demanding group at the Base used. One to the base. coupled sterically demanding Group may lead to obstruction of further synthesis. biotin, Digoxigenin and fluorescent dyes such as fluorescein, tetramethylrhodamine, Put Cy3 dye 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, p. 4513, Wiemann et al. Analytical Biochemistry 1996, v. 234, p. 166, Heer et al. BioTechniques 1994 16 p. 54). Although the polymerases can incorporate such NTs * at longer intervals, However, they have difficulty installing these NTs * one after the other.

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

Of the Difference to the 3'-OH Terminators method is that not a blockade of the for the synthesis necessary 3'-OH Desired group, but a knotted to the base group as a steric hindrance to the further installation is used. The 3'-OH group stays the same all the time free.

4.5.2 General structure of the NT *

Semiterminators can have different structures. Their common features are in 7a shown. This structure is characterized in that a steric group (D) and the fluorescence label (F) are bound to the base via a cleavable linker (AE).

When basis for the NTs * serve deoxynucleoside triphosphates with adenosine (A), guanosine (G), cytidine (C) and thymidine (T) as the nucleoside residue. Instead of Thymidine is preferably uridine used as a nucleoside residue. Instead of Inosine can be used by guanosine.

4.5.3 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 labeled with a marker (F) characteristic for it ( 7 ). 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 U.S. Patent 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. Patent 6130101). 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. Chr. 278 p. 363, Wagoner Methods in Enzymology 1995 v. Chr. 246 p. 362), s. Examples 1 and 2.

4.5.4 Nature of the steric demanding group

The group (D) ( 7 Biotin, digoxigenin, and fluorescent dyes are examples of such a bulky group (Zhu et al., Cytometry 1997, v. 28, p.206, Zhu et al NAR 1994, v. 22, pp. 3418, Gebeyehu et al., NAR 1987, v. 15, pp. 4513, Wiemann et al Analytical Biochemistry 1996, v. 234, p166, Heer et al., BioTechniques 1994 v. 16 p. 54). The chemical structure of this group is not limited provided that it does not significantly interfere with the incorporation of the labeled NT * to which it is attached and does not cause irreversible interference with the enzymatic reaction.

These Group can be self-employed Part in the linker (7a) occur or with the dye (7b) or the cleavable Be identical to group (7d). By the cleavage of the linker becomes this sterically demanding group (D) after the detection of the Signal removed, so that the Polymerase can incorporate another labeled NT *. At a Structure as in 7d becomes the steric group through the cleavage eliminated.

In a preferred embodiment, the fluorescent dye performs the function of such a sterically demanding group, so that a labeled nucleotide a in 7b having shown structure.

In another preferred embodiment, the photolabile cleavable group assumes the function of such a sterically demanding group ( 7d ).

4.5.5 Linker

The marker is preferably attached to the base via a spacer of different lengths; a so-called linker, bound. Examples of linkers are in 7e , f, h, i, j. Preferably, this linker is attached to the base at one of the sites that does not participate in base pairing. In the preferred case, the sites to which the linker is attached are the 5-position in the pyrimidine ring and the 7-position in the purine ring. Examples of the coupling of a linker to the base can be seen from the following sources (Hobbs et al., U.S. Patent 5,047,519, Khan et al., U.S. Patent 5,821,356, Hanna M. Method, Enzymology 1996, v. 274, p Zhu et al NAR 1994 v. 22 p 3418, Herman et al., Methods in Enzymology 1990 v. 184 p 584).

The entire length of the linker may vary. It corresponds to the number of carbon atoms in sections A, C, E and is preferably between 3 and 20. Optimally, it is between 4 and 10 atoms. The chemical composition of the linker (Sections A, C, E in 7a ) is not limited, provided that it remains stable under reaction conditions and does not cause any disturbance of the enzymatic reaction.

4.5.6 Splittable connection, cleavage

The linker carries a cleavable or cleavable group (B). This fissile connection he allows removal of the marker and steric obstruction at the end of each cycle. Their choice is not limited provided that it remains stable under the conditions of the enzymatic sequencing reaction, does not cause irreversible disruption of the polymerase and can be cleaved under mild conditions. By "mild conditions" are meant those conditions which do not destroy the NSKF-primer complex, for example the pH preferably being 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 calculated as Tm (NSKF primer complex) minus 5 ° C (eg Tm is 47 ° C, then the maximum temperature at 42 ° C, under these conditions are especially disulfide compounds and photolabile compounds as fissile compounds).

Preferably belongs said compound to be chemically or enzymatically cleavable or photolabile compounds. As examples of chemically cleavable Groups are preferably ester and disulfide compounds (Herman et al. Method in Enzymology 1990 v. Chr. 184 p.584, Lomant et al. J. Mol. Biol. 1976 v. Chr. 104 243, "Chemistry of carboxylic acid and esters "S. Patai 1969 Interscience Publ.). Examples of photolabile compounds can 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. Chr. 9 P. 225.

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 (Sigma) in the cleavage of a disulfide compound), see Example 1, or physically (e.g., by illuminating the surface with light of a certain wavelength for the cleavage of a photolabile Group).

After cleavage, a linker residue (A) remains at the base ( 7c ).

The Synthesis of a cleavable linker is shown by way of examples (cf. Examples 1 and 2).

4.5.7 Combination of Polymerase and NT *

Overall, the size, the charge and the chemical structure of the marker, the length of the cleavable linker and the linker residue as well as the choice of polymerase play an important role. Together, they determine whether the tagged NT * is incorporated into the growing nucleic acid chain by the polymerase and whether it prevents the next labeled NT * from being incorporated. Two conditions must be taken into account:
On the one hand, it is important that the polymerase can further extend the nucleic acid chain with the incorporated modified NT * after cleavage of the linker. So it is important that the linker residue "A" ( 7c ) after cleavage is not a significant disruption to further synthesis. On the other hand, built-in, non-split NTs * must be an obstacle. Many NTs * suitable for the reaction can be synthesized. More specifically, for each combination of polymerase and NTs *, a series of preliminary experiments must be performed in which the suitability of a specific NT * type for sequencing is tested.

The Buffer conditions are according to the polymerase manufacturer selected. The reaction temperature is for non-thermostable polymerases selected according to the manufacturer's instructions (e.g. 37 ° C for Sequenase Version 2), for thermostable polymerases (e.g., Taq polymerase) is the reaction temperature maximum temperature value (x). This temperature value (x) depends on the Tm of the NSKF primer complex and is termed Tm (NSKF primer complex) minus 5 ° C calculated (e.g., Tm is 47 ° C, then the maximum reaction temperature is 42 ° C). These Buffer conditions and reaction temperature are further described as "optimal buffer and temperature conditions ".

The Reaction time (corresponds to the duration of the installation step in a Cycle) preferably less than one hour, ideally the reaction time is between 10 sec and 10 min.

• a) NT* mit einem kurzen Linkerrest (Synthese siehe Beispiel 2, 7e, h, i): dNTP-SS-TRITC (L7), dNTP-SS-Cy3 (L11) in Kombination mit Sequenase Version 2, Taq-Polymerase (GibcoBRL), ProHA-DNA-Polymerase (Eurogentec) oder DNA-Polymerase Beta aus Säugetieren (Chimerx).
• b) NT* mit einem langen Linkerrest (Synthese siehe Beispiel 1, 7f, g, j): dNTP-SS-TRITC (L14) in Kombination mit Sequenase Version 2 oder oder ProHATM-Polymerase (Eurogentech).

As examples of suitable combinations between NT * and polymerase, the following combinations may be mentioned:

• a) NT * with a short linker residue (for synthesis see Example 2, 7e , h, i): dNTP-SS-TRITC (L7), dNTP-SS-Cy3 (L11) in combination with Sequenase version 2, Taq polymerase (GibcoBRL), ProHA DNA polymerase (Eurogentec) or DNA polymerase beta from mammals (Chimerx).
• b) NT * with a long linker residue (for synthesis see Example 1, 7f , g, j): dNTP-SS-TRITC (L14) in combination with Sequenase version 2 or ProHATM polymerase (Eurogentech).

The Suitability of a linker residue on the base (A) for the reaction can be in to test a test system. In this case, split NTs * are inserted successively into a nucleic acid chain. For example, one uses dUTP * with the desired cleaved linker residue, poly-dA as a template, oligo dT20 primer, the desired polymerase and carries out for the respective polymerase suitable optimal buffer and temperature conditions a reaction through. The NT * concentration is preferably between 5 μM and 200 μM. After the reaction, the number of nucleic acid into the chain built-in NTs *, e.g. by the separation of the length in a gel. For the conclusions the suitability of the linker residue can be used as follows: If the polymerase can incorporate more than 20 NTs *, it is Linkerrest for a sequencing reaction suitable. When installing less than 20 cleaved NTs * is this combination of NT * and polymerase not optimal for the sequencing reaction.

If a suitable linker residue is established, one must in another test system check, whether the marked, uncleaved NTs * function as semiterminators. That's being checked by selecting the marked NTs * under optimal for the reaction Buffering and temperature conditions with the polymerase and a template be incubated. The NT * concentration is preferably between 5 μM and 200 μM. The matrix is to be chosen that the Installation of multiple NTs * would be expected sequentially, e.g. For dUTP * you can use polydA, like use in the example above. Ideally, the builds Polymerase only a single NT * one.

If at given optimum buffer and temperature conditions a polymerase several NTs * can be installed one after the other the reaction parameters (e.g., NT * concentration, reaction temperature) change and the respective combination of polymerase and NT *. The most important thing is that the Polymerase in the given time (preferably between 10 sec and 10 min) does not install a second NT *.

According to the invention this adaptation in one embodiment through the change the reaction temperature. The other parameters of the reaction will be kept constant.

When Polymerases are used, for example, polymerases, the polymerases without 3'-5'-endonuclease activity, preferably from the group consisting of viral polymerases from Sequenase type, beta polymerases from eukaryotes, and thermostable polymerases. In particular, come Sequenase version 2, mammalian polymerase beta, Taq polymerase or ProHATM polymerase into consideration.

The NT * concentration in these experiments is usually between 5 μM and 200 μM, preferably between 10 and 100 μM. The concentration of the polymerase and the buffer conditions become low Information chosen by the manufacturer. The duration of the reaction can vary and is preferably between 10 sec and 10 min, which is the duration of the installation step (a) in one Cycle would correspond. For non-thermostable polymerases, e.g. Sequenase version 2 (Amersham Pharmacia Biotech), exonuclease free Klenow fragment of DNA Polymerase I (Amersham Pharmacia Biotech) becomes the reaction temperature from conventional 37 ° C preferably at 20 ° C up to 30 ° C reduced. For thermostable polymerases, e.g. Taq polymerase (GibcoBRL), ProHATM polymerase (Eurogentech) becomes the reaction temperature from conventional 70-75 ° C preferably reduced to values between 30 ° C and the temperature value (x). This temperature value (x) depends from the Tm of the NSKF primer complex and is calculated as Tm (NSKF primer complex) minus 5 ° C (e.g., Tm is 47 ° C, then the temperature value (x) is 42 ° C).

In another preferred embodiment The invention is carried out by adjusting the reaction conditions the reduction of the NT * concentration to below 5 μM, the others Parameters of the reaction (buffer conditions, temperature conditions) are kept constant. The concentration of NT * is preferably with this adjustment between 0.5 and 5 μM. The duration of the reaction is between 10 sec and 10 min. The most important in the choice of NT * concentration is that the Polymerase in the given time (preferably between 10 sec and 10 min) does not install a second NT *.

After optimizing the reaction conditions for the incorporation of a single NT * one must repeat the reaction with cleaved NTs *. Under appropriately changed reaction parameters must Polyme rase the split NTs * can install successively.

The Optimization reaction correlates with the installation step, step (b), in a cycle. The for the optimization reaction determined conditions, the temperature, the concentration of NT *, the buffer conditions, the duration of the reaction be for taken over the reaction on the surface.

Under These reaction conditions, the incorporation of NT * in the NSKFs preferably such that on more than 50% of the NSKFs involved in the sequencing reaction in one cycle a marked NT * is incorporated, preferably more than 90%. That depends together with that some nucleic acid chains. the reaction is very slow. An installation of the NTs * at each complementary position in each cycle is sought, but not required, because only the successful Installation reactions are detected and evaluated; a delayed reaction in the following cycle leads not a sequencing error.

Preferably is for all NTs * use the same polymerase. But it can also be different polymerases for different NTs * are used.

4.5.8 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.

4.6 detection apparatus

Separate molecules on a surface can be examined with different methods. There are several Method known: e.g. AtomForce Microscopy, Electron Microscopy, Near field fluorescence microscopy, wide field fluorescence microscopy, TIR microscopy etc. (Science 1999 v. 283 1667, Unger et al., BioTechniques 1999 v. 27 p. 1008, Ishijaima et al. Cell 1998 v. Chr. 92 p.161, Dickson et al. Science 1996 v. Chr. 274, p. 966, Xie et al. Science 1994 v. Chr. 265 p. 361, Nie et al. Science 1994 v. Chr. 266 p. 1018, Betzig et al. Science 1993 v. 262 p. 1422).

According to the invention Fluorescence signals of individual NTs incorporated in the nucleic acid chain * preferably with a wide-field fluorescence microscope or a laser scanning microscope or a TIRF microscope (Total Internal Reflection Fluorescence Microscope).

Various variants of the construction of such an apparatus are possible (Weston et al., J. Chem. Phys., 1998, 109, p.7474, Trabesinger et al., Anal Chem., 1999, 71, 279, Adachi et al., Journal of Microscopy 1999 v. 195 p.125, Unger et al BioTechniques 1999 v. 27 p 1008, Ishijaima et al Cell 1998 v. 92 p 161, Dickson et al Science 1996 v. 274 p 966, Tokunaga et Bichem, Biophys., Res. Com., 1997, pp 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 mic roscopy "1998. 2. Herman BIOS Scientific Publishers," Handbook of biological confocal microscopy "1995 J. Pawley Plenary Press) .Differences in their concrete structure arise from the variation of their individual parts.The device for the excitation light can eg based on Both CCD cameras and PMTs can be used for the detection device, for 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. Herman BIOS Scientific Publishers," Handbook of biological confocal microscopy "1995 J. Pawley Plenary Press.) It is not the task The basic structure of a suitable apparatus is shown in a diagram 8th explained. It consists of the following elements:
Light source for excitation of fluorescence (1)
Light conducting part (2)
Scan table (3)
Device for selecting spectra (4)
Detection device (5)
Computer with control and analysis functions (6)

These Elements of the apparatus can be acquired commercially (microscope companies: Zeiss, Leica, Nikon).

In the following, for example, a combination of these elements suitable for the detection of individual molecules is to be presented:
Wide field fluorescence microscope Axioplan 2 (Zeiss) with mercury vapor lamp
Lens Planneofluar 100x, NA 1.4 (Zeiss)
Camera Photometrix
Computer with software for control and analysis

following the procedure for the detection should be explained. Please note the general rules of fluorescence microscopy ("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).

• 1) Vorbereitung zur Detektion
• 2) Durchführung eines Detektionsschrittes in jedem Zyklus, wobei jeder Detektionsschritt als Scanvorgang abläuft und folgende Operationen umfaßt: a) Einstellung der Position des Objektivs (X,Y-Achse), b) Einstellung der Fokusebene (Z-Achse), c) Detektion der Signale einzelner Moleküle, Zuordnung des Signals zu NT* und Zuordnung des Signals zum jeweiligen NSKF, d) Verschiebung zur nächsten Position auf der Oberfläche.

The detection comprises the following phases:

• 1) Preparation for detection
• 2) carrying out a detection step in each cycle, each detection step being carried out as a scanning operation and comprising the following operations: a) adjustment of the position of the objective (X, Y-axis), b) adjustment of the focal plane (Z-axis), c) detection of the Signals of single molecules, assignment of the signal to NT * and assignment of the signal to the respective NSKF, d) shift to the next position on the surface.

The signals from NTs * built into the NSKFs are registered by scanning the surface. Scanning may be accomplished in a variety of ways ("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. Herman BIOS Scientific Publishers, "Handbook of biological confocal microscopy" 1995 J. Pawley Plenary Press). Preferably, a discontinuous scan is selected. The lens is moved stepwise over the surface ( 8a ), so that a two-dimensional image (2D image) arises from each surface position ( 8b , c).

This 2D image can be created by various methods: e.g. by the laser scan of a position of the microscope field (laser scanning microscopy) or by a camera recording at a position (see manuals of the Microscopy). As an example, the detection of single molecules with a CCD camera described.

The Detection is explained using the example of sequencing a 1-Mb DNA piece:

1) Preparation for detection:

At the Beginning determines how many NSKFs to reconstruct the original Sequence must be analyzed. In the case of a reconstruction according to the shotgun method ("Automated DNA sequencing and analysis "p. 231 ff. 1994 M. Adams et al. Academic Press, Huang et al. genome Res. 1999 v. Chr. 9 p. 868, Huang Genomics 1996 v. Chr. 33 p. 21, Bonfield et al. NAR 1995 v. 23 p. 4992, Miller et al. J. Comput. Biol. 1994 v. 1 p. 257) the following factors play a role: 1) Of each NSKF will sequencing a sequence of about 300-500 NTs certainly. 2) The total length the sequence to be analyzed is important. 3) In the sequencing has to be certain degree Redundancy can be achieved to increase accuracy and eventuality Correct mistakes. Overall, to the reconstruction of the largest part the original one Sequence requires about 10 to 100 times the amount of crude sequences, i.e. in this example with an Mb, 10 to 100 Mb becomes raw sequence data second hand. With an average sequence length of 400 bp per NSKF one needs corresponding to 25,000 to 250,000 DNA fragments.

2) implementation of a Detection step in each cycle

to Need sequencing The positions of the NSKFs are determined to provide a basis for assignment has the signals. The knowledge of these positions allows a statement about that, whether the signals of individual molecules come from built-in NTs * or from randomly bound to the surface NTs *. These positions can be identified with different methods.

In a preferred embodiment The positions of immobilized NSKF are identified during sequencing. The fact is used that the signals of the in the Nucleic acid chain installed NTs * always have the same coordinates. That's because of the fixation the nucleic acid chains guaranteed. The nonspecifically bound NTs * bind randomly at different sites the surface.

to Identifying the positions of fixed NSKFs will be the signals on agreement checked their coordinates from several consecutive cycles. The can e.g. done at the beginning of the sequencing. The matching Coordinates are evaluated and stored as coordinates of the DNA fragments.

The Scan system must be reproducible over several Cycles the surface can scan. X, Y and Z-axis settings at each surface position can be from controlled by a computer. Stability and reproducibility of the Setting lens positions in each scan decide on the quality the detection and thus over the identification of the signals of individual molecules.

a.) Setting the position of the lens (X, Y axis)

The mechanical instability the commercially available Scant and the low reproducibility of repeated adjustment the same X, Y positions make a precise analysis of the signals of individual Molecules over several Cycles difficult. There are many possibilities, a match the coordinates in repeated settings to improve or possible To control deviations. As an example, a check option cited. After a rough mechanical adjustment of the lens position becomes a control image of a pattern firmly attached to the surface made. Even if the mechanical adjustment is not exactly the same Coordinates (deviations of up to 10 μm are possible), you can make a correction by means of optical control. The control picture of the pattern serves as a coordinate system for the image with signals from built-in NTs *. A prerequisite for such a correction is that no further movements of the surface be made between these two shots. Signals from individual molecules are set in relation to the pattern so that an X, Y deviation in the Pattern position same X, Y deviation in the position of the signals of individual molecules. The control picture from the pattern can before, during or after the detection of individual molecules. Such Control image must be correspondingly be made on a new surface position at each setting.

b) Setting the focal plane (Z-axis)

The surface is not absolutely flat and has various bumps. This is changing the surface-to-lens distance when scanning neighboring bodies. These differences in distance can do this to lead, that individual molecules leave the focal plane and thus escape the detection.

Out For this reason, it is important that when Scan the surface a reproducible adjustment of the focal plane at each lens position is reached.

It are different ways to adjust the focal plane reproducibly. For example, can The following method can be used: Since the excitation of individual molecules to extinguish their Lead to fluorescence can, will on the surface a marker applied, which serves to adjust the focal plane. Thereafter, the detection of the signals of individual molecules. Of the Marker can be of any nature (e.g., dye or pattern), but must not interfere with the detection and the reaction.

c) detection of the signals single molecules, Assignment of the signal to NT * and assignment of the signal to the respective one NACF.

• 1. Durch Verwendung von geeigneten Filtern (Zeiss-Filtersätze) wird für jeden Fluoreszenzkanal ein Grauwertbild erzeugt.
• 2. Aus einem aufgenommenen Mehrkanal-Farb-Bild werden mit Hilfe eines geeigneten Algorithmus durch ein Bildverarbeitungsprogramm die relevanten Farbkanäle extrahiert und jeweils als Grauwertbild einzeln weiterverarbeitet. Zur Kanalextraktion wird dabei ein für den jeweiligen Kanal spezifischer Farb-Schwellwertalgorithmus eingesetzt. So entstehen zunächst aus einem Mehrkanal-Farbbild einzelne Grauwertbilder 1 bis N. Diese Bilder definieren sich wie folgt: GB_N = (s(x,y)) einkanaliges Grauwertbild N = {1, ..., Anzahl der Fluoreszenzkanäle}. M = {0,1, ..., 255} Grauwertmenge S = (s(x,y)) Bildmatrix des Grauwertbildes x = 0, 1, ..., L-1 Bildzeilen y = 0, 1, ..., R-1 Bildspalten (x,y) Ortskoordinaten eines Bildpunktes s(x,y) ∊ M Grauwert des Bildpunktes.

The two-dimensional image of the reaction surface generated with the aid of the detection system contains the signal information of NT * s incorporated in the NSKFs. These must be extracted from the total data volume of the image information with suitable methods before further processing. The necessary algorithms for scaling, transforming and filtering the image information belong to the standard repertoire of digital image processing and pattern recognition (Haberäcker P. "Practice of Digital Image Processing and Pattern Recognition." Hanser-Verlag, Munich, Vienna, 1995; Galbiati LJ "Machine vision and digital image processing fundamentals. "Prentice Hall, Englewood Cliffs, New Jersey, 1990). The signal extraction preferably takes place via a gray scale image, which images the brightness distribution of the reaction surface for the respective fluorescence channel. If several nucleotides with different fluorescent dyes are used in the sequencing reaction, a separate gray scale image can first be generated for each fluorescently labeled nucleotide (A, T, C, G or U) used. In principle, 2 methods can be used:

• 1. By using suitable filters (Zeiss filter sets), a halftone image is generated for each fluorescence channel.
• 2. From a recorded multichannel color image, the relevant color channels are extracted by means of a suitable algorithm by an image processing program and further processed individually as a gray value image. For channel extraction, a color threshold algorithm specific to the respective channel is used. Thus, first of all, gray-scale images 1 to N result from a multichannel color image. These images are defined as follows: GB _N = (s (x, y)) single-channel gray value image N = {1,..., Number of fluorescence channels}. M = {0,1, ..., 255} Gray value set S = (s (x, y)) Image matrix of the gray scale image x = 0, 1, ..., L-1 Image lines y = 0, 1, ... , R-1 image columns (x, y) spatial coordinates of a pixel s (x, y) ε M gray value of the pixel.

• I. Vorverarbeitung des Bildes, so zum Beispiel gegebenenfalls Reduktion des durch die Digitalisierung der Bildinformation entstandenen Bildrauschens, etwa durch Grauwertglättung.
• II. Prüfung jedes Bildpunkt (x,y) des Grauwertbildes, ob dieser Punkt im Zusammenhang mit den ihn umgebenden unmittelbaren und weiter entfernten Nachbarbildpunkten die Eigenschaften eines Fluoreszenzpunktes erfüllt. Diese Eigenschaften hängen unter anderem von der verwendeten Detektionsapparatur und der Auflösung des Grauwertbildes ab. Sie können beispielsweise ein typisches Verteilungsmuster von Helligkeits-Intensitätswerten über einer den Bildpunkt umgebenden Matrix darstellen. Die dazu verwendbaren Methoden der Bildsegmentierung reichen von einfachen Schwellwertverfahren bis hin zur Verwendung neuronaler Netze.

From this data set, the relevant image information is now extracted by a suitable program. Such a program should realize the following steps:
Perform for GB ₁ to GB _N :

• I. Pre-processing of the image, so for example, if necessary, reduction of the image noise resulting from the digitization of the image information, such as gray scale smoothing.
• II. Examination of each pixel (x, y) of the gray value image, whether this point fulfills the properties of a fluorescence spot in connection with the immediate and more distant neighboring pixels surrounding it. Among other things, these properties depend on the detection apparatus used and the resolution of the gray value image. For example, they can represent a typical distribution pattern of brightness intensity values over a matrix surrounding the pixel. The methods of image segmentation that can be used range from simple threshold procedures to the use of neural networks.

Meets one Pixel (x, y) these requirements, then follows a comparison with the coordinates of previously performed sequencing cycles identified NSKFs. If there is a match, the Assignment of the signal with that from the respective fluorescence channel resulting nucleotide to this NSKF. Signals with mismatched Coordinates are scored as background signals and discarded. The analysis of the signals can be carried out parallel to the scanning process.

In an exemplary embodiment, an 8-bit gray-scale image with a resolution of 1317 × 1035 was used Pixel used. In order to reduce the changes in the image caused by digitization, the entire image was first preprocessed: each pixel was assigned the average brightness of its 8 neighbors. At the selected resolution, this produces a typical pattern for a fluorescence spot of a central pixel with the greatest brightness value and neighboring pixels with brightnesses that decrease in all directions. If one pixel fulfilled these criteria and the centrifugal brightness decrease exceeded a certain threshold (to exclude weak fluorescence spots), then this central pixel was considered to be a coordinate of a fluorescence spot.

d) displacement of the lens to the next Position on the surface. After the detection of the signals of individual molecules, the lens is above a other position of the surface positioned.

All in all For example, a sequence of recordings with the control of X, Y position, focal plane adjustment and detection single molecules be done with every new lens position. These steps can be controlled by a computer.

4.7 Timing the process steps

The scanning process and the biochemical reaction take some time. By switching these processes one after another, one can achieve optimal performance of the apparatus. In a preferred embodiment, the reaction is carried out on two separate surfaces ( 9 ).

When Example can be a surface with immobilized NSKFs in 2 spatially isolated parts are separated so that reactions to these two Share independently can run away from each other. In another example NSKFs also immobilized from the outset on 2 separate surfaces become.

After that the reaction is started. The principle is that while on a part of the surface the Reaction and washing steps take place, the second part is scanned becomes. This allows one to have a continuous process of analysis achieve and increase the speed of sequencing.

The Number of surfaces, on which the reaction takes place, can also be bigger than Be 2. This makes sense if the reaction is timed limiting step occurs, i. the detection of the signals the surface faster than the reaction and washing steps. Around adjust the total duration of the reaction to the detection duration, Every single step of the reaction can be on a single surface with a time delay compared to the next surface expire.

The The invention is illustrated below by way of examples.

Examples

Example 1:

Modified dUTP with a long cleavable linker ( 7f-1 ) Starting materials are 5- (3-aminoallyl) -2'-deoxyuridine-5'-triphosphate, AA-dUTP, (Sigma), 3,3'-dithio-bis (propionic acid N-hydroxysuccinimide ester), DTBP-NHS, (Sigma), 2-mercaproethanolamine, MEA, (Sigma). To 100 μl of 50 mM solution of AA-dUTP in 100 mM borate buffer pH 8.5, 3 equivalents of DTBP-NHS in DMF (25 μl 0.4 M solution) are added. The reaction mixture is at RT for 4 hours. incubated. Subsequently, conc. Ammonium acetate solution (pH 9) is added until the total concentration of CH ₃ COONH ₄ in the reaction solution is 100 mM, and the reaction is incubated for an additional hour. Thereafter, 200 .mu.l 1 M MEA solution, pH 9, are added to this mixture and incubated for one hour at RT. Subsequently, a saturated solution of I ₂ in 0.3 M KI solution is added dropwise to this mixture until the iodine color persists. The modified nucleotides are separated on a DEAE cellulose column in ammonium carbonate gradient (pH 8.5) from other reaction products. Isolation of the nucleotide with the cleavable linker is carried out on RP-HPLC. Dyes can now be coupled to this linker by various methods ("Handbook of Fluorescent Probes and Research Chemicals" 6th ed. 1996, R. Haugland, Molecular Probes, Wagoner Method in Enzymology 1995 v. 246, p.362, Jameson et al. Method in Enzymology 1997, v. 278, p. 363).

Other nucleotide analogues (for example, according to Hobbs et al., US Patent 5,047,519, Khan et al., US Patent 5,821,356) can be used in the reaction, so that nucleotide analogues having structures in 7f-2 , 3, 4 and 7g-1 , 2 can be generated.

As an example of the coupling of a dye to the linker, the coupling of TRITC (tetramethylrhodamine isothiocyanate) is indicated (NT * structure 7y )

The modified with the cleavable linker dNTP (300 nmol) is in 30 ul 100 mM Sodium borate buffer pH 9 dissolved (10 mM NT *). This will be 10 ul 10 mM TRITC in dimethylformamide (DMF) and incubated for 4 h at RT. The dye modified NT * is purified by RP-HPLC in a methanol-water gradient.

example the cleavage of the disulphide compound in the modified NT *. The split is done by adding 20 to 50 mM dithiothreitol solution DTT (Sigma) solution pH 8 on the reaction surface. The surface will be 10 min. with this solution incubated, then the solution removed and the surface with a buffer solution washed to remove DTT residues.

Example 2

Modified dUTP (dUTP-SS-CH ₂ CH ₂ NH ₂ ) with a short cleavable linker ( 7e-1 ). The starting substances used are: bis-dUTP, synthesized according to Hanna (Method in Enzymology 1989, v. 180, p. 383), 2-mercaptoethanolamine (MEA) (Sigma).

100 μl of 100 mM MEA solution pH 8.5 in H ₂ O are added to 400 μl of 100 mM Bis-dUTP in 40 mM borate buffer pH 8.5 and incubated for 1 hour at RT. Subsequently, a saturated solution of I ₂ in 0.3 M KI solution is added dropwise to this mixture until the iodine color persists. The nucleotides (bis-dUTP and dUTP-SS-CH ₂ CH ₂ NH ₂ ) can be separated from other reaction products, for example by ethanol precipitation or on a DEAE cellulose column in the ammonium carbonate gradient (pH 8.5). Bis-dUTP does not interfere with the subsequent coupling of a dye to the amino group of the linker, so that the separation of the dUTP-SS-CH ₂ CH ₂ NH ₂ from bis-dUTP can take place in the final purification step.

In a similar way, dCTP ( 7-e2 ), whereby bis-dCTP serves as the starting substance (synthesized according to Hanna et al., Nucleic Acid Research 1993, v. 21, p.2073).

At the linker can now dyes can be coupled with different methods ("Handbook of Fluorescent Probes and Research Chemicals "6th ed. 1996, R. Haugland, Molecular Probes, Wagoner Method in Enzymology 1995 v. 246, p. 362, Jameson et al. Method in Enzymology 1997, v. Chr. 278, p. 363).

As an example of the coupling of a dye to the linker, the coupling of the FluoroLink ^™ Cy3 monofunctional dye (Amersham Pharmacia biotech) (NT * structure 7i ). This is a monofunctional NHS ester fluorescent dye. The reaction is carried out according to the manufacturer's instructions:
The cleavable linker-modified dNTP (300 nmol) is dissolved in 300 μl of 100 mM sodium borate buffer pH 8.5. For this purpose, dye (300 nmol) is added and incubated for 1 h at RT. The dye-modified NT * is purified by RP-HPLC in a methanol-water gradient.

Another example of the coupling of a dye to the linker is the coupling of TRITC (tetramethylrhodamine-5-isothiocyanate, Molecular Probes) (dUTP-SS-TRITC 7h ).

The modified with the cleavable linker dNTP (300 nmol) is in 30 ul 100 mM Sodium borate buffer pH 9 dissolved (10 mM NT *). This will be 10 ul 10 mM TRITC in DMF and incubated for 4 h at RT. The cleaning of the modified with the dye NT * via RP-HPLC in a methanol-water Gradient.

Example 3:

Sequence analysis with 4 marked NTs *

at a preferred embodiment all four NTs used in the reaction are labeled with fluorescent dyes.

3A.

Reconstruction of the original sequences according to the shotgun principle (Automated DNA sequencing and analysis, page 231 et seq., 1994, M. Adams et al., Academic Press, Huang et al., Genom Res., 1999, v. 9 P. 868, Huang Genomics 1996 BC. 33 p. 21, Bonfield et al. NAR 1995 v. 23 p. 4992, Miller et al. J. Comput. Biol. 1994 v. Chr. 1 p. 257). (This principle is particularly useful in the analysis of new, unknown sequences.)

3A-1 Sequencing of a long piece of DNA ( 1 )

In the following, the sequencing of a 1 Mb long piece of DNA will schematically illustrate the sequencing of long nucleic acid chains ( 1 ). The sequencing is based on the shotgun principle ("Automated DNA sequencing and analysis", page 231 et seq., 1994, M. Adams et al., Academic Press, Huang et al., Gen. Res., 1999, 9, 868, Huang Genomics, 1996, p 33, p.21, Bonfield et al., NAR 1995, v. 23, p.4992, Miller et al., J. Comput., Biol., 1994, v. 1, p. The material to be analyzed is prepared for the sequencing reaction by breaking it down into fragments of preferably 50 to 1000 bp in length. Each fragment is then provided with a primer binding site and a primer. This mixture of different DNA fragments is now fixed on a flat surface. The unbound DNA fragments are removed by a washing step. Thereafter, the sequencing reaction is carried out on the entire reaction surface. To reconstruct a 1 Mb DNA sequence, the sequences of NSKFs should preferably be longer than 300 NTs, on average about 400 bp.

There Only one marked NT * is installed per cycle, are at least 400 cycles necessary for sequencing.

All in all is for the reconstruction of the original one Sequence requires about 10 to 100 times the amount of crude sequences, i.e. 10 to 100 Mb. With an average sequence length of about 400 bp per NSKF needed correspondingly 25,000 to 250,000 DNA fragments, by more than 99.995% to cover the entire sequence.

The detected NSKF sequences represent a population of overlapping Partial sequences dealing with commercially available programs to the overall sequence put together the NSK ("Automated DNA sequencing and analysis "S. 231 ff. 1994 M. Adams et al. Academic Press, Huang et al. Genome Res. 1999 v. 9 p. 868, Huang Genomics 1996 v. Chr. 33 p. 21, Bonfield et al. NAR 1995 v. 23 p. 4992, Miller et al. J. Comput. Biol. 1994 v. Chr. 1 P. 257).

3A-2 Sequencing of Gene products using the example of cDNA sequencing

In a preferred embodiment can Instead of a sequence, several sequences were analyzed in one approach become. The originals Sequences can from the obtained raw data e.g. after the shotgun principle be reconstructed.

First, be NSKFs generated. One can e.g. Convert mRNA into a double-stranded cDNA and this Fragment cDNA with ultrasound. Subsequently, these NSKFs with provided a primer binding site, denatured, immobilized and hybridized with a primer. Please note this variant the sample preparation that the cDNA molecules incomplete mRNA sequences can represent (Method in Enzymology 1999, v. 303, p. 19 and other articles in this volume, "cDNA library protocols "1997 Humana Press).

A different possibility in the generation of single-stranded NSKFs of mRNA is involved in the reverse transcription of mRNA randomized primers. There are many relatively short antisense DNA fragments (Zhang-J et al., Biochem., J., 1999, v., 337, p. 231, Ledbetter et al. J. Biol. Chem. 1994 v. Chem. 269 p. 31544, Kolls et al. Anal. Biochem. 1993 v. 208 p. 264, decraene et l1. Biotechniques 1999 v. Chr. 27 p. 962). These fragments can subsequently be provided with a primer binding site (s.o). Further steps correspond to the procedures described above. With this method can complete mRNA sequences (from the 5 ' until the 3'-end) be analyzed as the randomized primers across the entire length of the mRNA tie.

immobilized NSKFs are analyzed by one of the above sequencing embodiments. Because mRNA sequences have significantly fewer repetitive sequences than e.g. genomic DNA, the number of detected signals of the built-in NTs * of a NSKF is less than 300 and is preferably between 20 and 1000. The number of NSKFs that need to be analyzed is calculated following the same principles as a shotgun reconstruction a long sequence.

NSKF sequences become the original ones according to the principles of the shotgun method Reconstructed gene sequences.

These Method allows the simultaneous sequencing of many mRNAs without prior cloning.

3B. Analysis of sequence variants

The confirmation an already known sequence or the detection of variants this sequence puts much less demands on length and Redundancy of the ascertained NSKFs. The sequence processing is easier in this case. The full sequence does not need to be reconstructed too become. The NSKF sequences are rather using a commercial available Program assigned to the full sequence and possible deviations detected. Such a program may e.g. BLAST or FASTA algorithm underlying ("Introduction to computational Biology "1995 M. S. Waterman Chapman & Hall).

The sequence to be analyzed is in NSKFs with one of the above Shared method. These NSKFs are obtained by the method according to the invention sequenced. Subsequently The detected sequences of NSKFs are not by the shotgun method but compared with the reference sequence and on assigned this way to their positions in the full sequence. there they may be genomic or cDNA sequences.

in the Contrary to a reconstruction after the shotgun method needs one for one the analysis of a sequence variant significantly less crude sequence data. So that can be 5 to 10 times Raw sequence quantity sufficient for the restoration of a new variant of a full sequence. With the shotgun method is for a restore a 10 to 100 times the amount of raw sequences needed ("Automated DNA sequencing and analysis "S. 231 ff. 1994 M. Adams et al. Academic Press, Huang et al. genome Res. 1999 v. Chr. 9 p. 868, Huang Genomics 1996 v. Chr. 33 p. 21, Bonfield et al. NAR 1995 v. 23 p. 4992, Miller et al. J. Comput. Biol. 1994 v. 1 p. 257).

The Length of determined NSKF sequences intended for unambiguous assignment sufficient for a given position in the reference sequence be, so can For example, already sequences with a length of 20 NTs (for example, not from repetitive sections in the human genome) become. For the comparative analysis of the repetitive sections become longer sequences needed. The exact length the sequences depends thereby from the task from. Preferably, the length of the determined NSKF sequences in the analysis of non-repetitive sections more than 20 NTs. For the analysis of the repetitive sections is preferably more than 500 NTs.

The Objectives in the sequencing of new variants of an already known full sequence can be very different. Mostly a comparison of the newly determined Sequence sought with the known full sequence / reference sequence. It can the two sequences from evolutionary different far apart Species originate. Various parameters of the composition of this both sequences can be compared. Examples of such an analysis are: Mutation or polymorphism analyzes and the analysis of alternative spliced Gene products.

following By way of example, a comparison of the sequence to be examined with a reference sequence without prior reconstruction of the analyte Sequence can be considered. Such a comparison may e.g. to the mutation or SNP analysis.

3B-1

A long sequence to be analyzed, eg 1 Mb, is divided into NSKFs using one of the above methods. These NSKFs are sequenced using the method according to the invention. The determined sequences of each individual NSKF are compared directly with the reference sequence. The reference sequence serves as the basis for the assignment of determined NSKF sequences, so that the complex reconstruction according to the shotgun method is omitted. Preferably, the length of the detected NSKF sequences in the analysis of non-repetitive sections is more than 20 NTs. For the analysis of the repetitive sections, it is preferably more than 500 NTs. The number of NSKFs to be analyzed depends on the total length of the sequence to be examined, the average length of the NSKF sequences and the necessary accuracy of the sequencing. With an average length of the determined NSKF sequence of 100 NTs, a total length of the sequence to be examined of 1 Mb and an accuracy which corresponds to the raw sequence determination (ie each site should preferably only be sequenced once) one needs, for example, about 5-fold Amount of raw sequences, ie 5 Mb, because the distribution of NSKFs over the Total sequence happens at random. In total, 50,000 NSKFs need to be analyzed to cover more than 99% of the total distance.

Then be the determined NSKF sequences using a commercially available Program assigned to the full sequence and possible deviations detected. Such a program may e.g. BLAST or FASTA algorithm underlying ("Introduction to computational Biology "1995 M. S. Waterman Chapman & Hall).

Example 4:

Sequence analysis with 2 marked NTs * and 2 unmarked NTs (2NTs * / 2NTs method).

In another embodiment be for the analysis of the sequences 2 modified NTs * and 2 unmodified NTs used.

These Method is particularly suitable for analyzing sequence variants (e.g. SNP or mutation analysis) and sets the knowledge of a reference sequence ahead. The full sequence is not reconstructed, but the sequences determined are determined by means of a program of the reference sequence assigned and possible deviations registered. Such a Program can e.g. based on the BLAST or FASTA algorithm ("Introduction to computational biology "1995 M. S. Waterman Chapman & Hall).

This embodiment is based on the principle that a sequence of 2 signals (labeled NT * s) can contain enough information to identify a sequence. The determined sequence is compared with the reference sequence and assigned to a specific position, eg:

The unknown variant of the reference sequence to be analyzed will be like described above for sequencing (NSK is transferred to NSKFs, this are ligated with PBS, then hybridized with a primer and on reaction surface immobilized). This way, NSKFs prepared with 2NTs * / 2NTs method sequenced. You get NSKF sequences, each NSKF sequence representing a sequence of 2NTs *. To a clear assignment of the determined sequence to a known To allow reference sequence must this Be long enough for the sequence. Preferably, the length of the detected NSKF sequences is more than 40 NT * s. Since 2 marked NTs * represent only part of the sequence, is the total length of the synthesized complementary Strand approximately twice as long as the sequence of detected NTs * (at 40 detected NTs * is the total length e.g. average of 80 NTs).

to Synthesis of a complementary Stranges require 4 nucleotides. As marked with a fluorescent dye NTs * in the present Invention as semiterminators, i. the termination exclusively at Availability modified NTs * occurs unmodified NTs in an additional Step in each cycle to be added to the reaction. The exact The position of this step in the cycle can vary. Important it is that the marked NTs * and the unmodified NTs are used separately.

• a) Zugabe einer Lösung mit modifizierten NTs* und Polymerasen auf die Oberfläche mit den bereitgestellten NSKFs
• b) Inkubation der immobilisierten Nukleinsäureketten mit dieser Lösung unter Bedingungen, die zur Verlängerung der komplementären Stränge um ein NT geeignet sind
• c) Waschen
• d) Detektion der Signale von einzelnen, modifizierten und in die den NSKFs komplementären neusynthetisierten Strängen eingebauten NTs*-Molekülen
• e) Entfernung der Markierung und der terminierenden Gruppe bei den eingebauten Nukleotiden
• f) Waschen
• g) Zugabe von 2 unmodifizierten NTs und Polymerasen
• h) Waschen.

A cycle in this embodiment may look like this, for example:

• a) Addition of a solution with modified NTs * and polymerases to the surface with the provided NSKFs
• b) incubation of the immobilized nucleic acid chains with this solution under conditions suitable for extension of the complementary strands around an NT
• c) washing
• d) Detection of the signals of individual, modified and incorporated into the NSKFs complementary Neusynthetisierten strands NTs * molecules
• e) Removal of the label and the terminating group in the incorporated nucleotides
• f) washing
• g) addition of 2 unmodified NTs and polymerases
• h) washing.

The Concentration of the NTs is preferably below 1 mM, ideally below 10 μM.

• 1) Die genetische Information in jedem der beiden komplementären DNA-Stränge ist identisch, so daß fehlende Informationen in einem Strang durch die Information aus dem anderen Strang vervollständingt werden können.
• 2) Durch bestimmte Paarkombinationen markierter NTs* kann man mit nur 2 NTs* die komplette Information aus einer doppelsträngigen DNA erhalten. Zulässige Kombinationen von NT*s bei dieser Ausführungsform sind: A*C*; A*G*; C*T*/C*U*; G*T*/G*U*. Bevorzugt wird die Kombination C* und U*.
• 3) Als Grundlage der Analyse dient eine bereits bekannte Referenzsequenz.
• 4) Die NSKFs stammen von beiden Strängen der zu analysierenden NSK und die ermittelten NSKF-Sequenzen decken die gesamte Länge der zu analysierenden Sequenz ab.

This 2NT * s / 2NTs method is useful, for example, for the SNP analysis of a genomic stretch of a gene or for double-stranded cDNA analysis. It is based on the following principles:

• 1) The genetic information in each of the two complementary DNA strands is identical, so that missing information in one strand can be completed by the information from the other strand.
• 2) By pair combinations of labeled NTs *, complete information can be obtained from a double-stranded DNA with only 2 NTs *. Permissible combinations of NT * s in this embodiment are: A * C *; A * G *; C * T * / C * U *; G * T * / G * U *. The combination C * and U * is preferred.
• 3) The basis of the analysis is an already known reference sequence.
• 4) The NSKFs are derived from both strands of the NSK to be analyzed and the determined NSKF sequences cover the entire length of the sequence to be analyzed.

At the the following example is explained like the information from a double-stranded DNA fragment with only 2 marked NTs * is won and how the differences to the original or unmutated sequence (reference sequence / comparison sequence) can be determined. Sequences under (1) and (2) are identical except for one place (underlined). A * and C * are marked.

1) sequence to be tested:

The sequence to be tested is sequenced by 2NT * s / 2NTs method, resulting in a population of NSKF sequences (detected NSKF sequences (n)). These detected NSKF sequences contain information from each strand:

2) Comparison sequence:

For analysis, a comparison sequence (reference sequence) is required:

3) Comparison sequence with matched determined NSKF sequences:

Using a program, determined NSKF sequences are assigned to specific points in the comparison sequence and possible deviations are detected:

With this embodiment, one can examine a double-stranded nucleic acid for SNP or mutations. The determined NSKF sequences are compared with a reference sequence. The basic rules of the comparison of a partial sequence and a complete sequence in the analysis with only 2 labeled NTs do not differ in principle from those which apply when comparing the sequences on the basis of all 4 marked NTs *. For details see Sequence comparison in mutation analysis and SNP analysis with 4NTs * (Example 3B).

SEQUENCE LISTING

Claims (43)

A method for the highly parallel sequence analysis of nucleic acid sequences or nucleic acid chains (NSKs), in which Generating fragments (NSKFs) of single stranded NSKs of about 50 to 1000 nucleotides in length which are overlapping partial sequences of the total sequences, one randomly assembles the NSKFs with a single primer or several different primers in the form of NSKF primer hybridization products on a reaction surface one carries out a cyclic assembly reaction of the complementary strand of NSKFs using one or more primers and one or more polymerases by a) adding to the immobilized NSKFs a solution containing one or more polymerases and one to four modified nucleotides (NTs * ), which are labeled with fluorescent dyes, wherein the simultaneous use of at least two NTs * each located on the NTs * fluorescent dyes are selected so that the NTs used * can be distinguished by measuring different fluorescence signals from each other, the N Ts * are structurally modified at the base so that the polymerase after incorporation of such NT * in a growing complementary strand is not able to incorporate another NT * in the same strand, the fluorescent dye is cleavable and the structural modification of a cleavable sterically demanding ligand, b) the stationary phase obtained in step a) are incubated under conditions suitable for extending the complementary strands, the complementary strands being extended by one NT * each, c) obtained in step b) washing stationary phase under conditions suitable for the removal of NTs * not incorporated in a complementary strand, d) detecting the individual NTs * incorporated in complementary strands by measuring the signal characteristic of the respective fluorescent dye, simultaneously determining the relative position of the NTs * individual fluorescence signals on the reaction surface (e) to generate unlabelled NTs or NSKFs, cleaving the fluorescent dyes and bulky ligands from the NTs * attached to the complementary strand, f) washing the stationary phase obtained in step e) under conditions effective to remove the fluorescent dyes and the ligands are suitable, the steps a) to f) are optionally repeated several times, wherein the relative position of individual NSKFs on the reaction surface and the sequence of these NSKFs by specific assignment of the fluorescence signals detected in step d) in successive cycles at the respective positions determine the NTs and one of the determined overlapping partial sequences, the total sequence of NSKs be true. Process according to Claim 1, characterized in that the NSKFs are bound to a reaction surface at a density of 10 to 100 NSKFs per 100 μm ² (stationary phase). Method according to claim 1, characterized in that that he the steps a) to f) of the cyclic synthesis reaction are repeated several times, whereby in each cycle only one marked NT * is used. Method according to claim 1, characterized in that that he the steps a) to f) of the cyclic synthesis reaction are repeated several times, in each case two different marked in each cycle NTs * uses. Method according to claim 1, characterized in that that he the steps a) to f) of the cyclic synthesis reaction are repeated several times, in each cycle four are marked differently NTs * uses. Method according to claim 1, characterized in that that the NSKs variants of a known reference sequence are and you the Steps a) to f) of the cyclic synthesis reaction are repeated several times, whereby in the cycles alternately two different marked NTs * and two unmarked NTs are used and the overall sequences determined by comparison with the reference sequence. Process according to claims 1 to 5, characterized that he in each of the NSKFs introduces 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. Process according to claims 1 to 7, characterized marked, that he 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 the NSKF primer hybridization products formed subsequently on the reaction surface binds. Process according to claims 1 to 7, characterized that he 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 which forms NSKF primer hybridization products, where the primers are mutually identical or different sequences exhibit. Process according to claims 1 to 7, characterized that he the primer first on the reaction surface immobilized and only afterwards interact with NSKFs under conditions conducive to hybridization the primer to the primer binding sites (PBSs) of the NSKFs suitable which causes NSKFs to surface and NSKF primer hybridization products are formed, wherein the primers with each other the same or different Have sequences. Process according to claims 1 to 10, characterized that the used for structural modification sterically demanding Ligand of the fluorescent dye used for labeling. Process according to claims 1 to 11, characterized that the reaction surface from the group consisting of silicone, glass, ceramics, plastics, Gels selected is. Method according to claim 12, characterized in that that the plastics Polycarbonates or polystyrenes are. Method according to claim 12, characterized in that that the Gels are agarose or polyacrylamide gels. Method according to claim 14, characterized in that that the Gels are 1 to 2% agarose gels or 10 to 15% polyacrylamide gels. Process according to claims 1 to 15, characterized that the DNA polymerase is a DNA polymerase without 3'-5 'endonuclease activity. Method according to claim 16, characterized in that that the Polymerase from the group consisting of Sequenase-type viral DNA polymerases, Beta polymerases from eukaryotes and thermostable polymerases selected is. Method according to claim 17, characterized in that that the DNA Polymerase Sequenase Version 2, Polymerase Beta from Mammals, Taq polymerase or ProHA DNA polymerase. Method according to claim 17, characterized in that that the DNA Polymerase Sequenase Version 2 is. Method according to claim 17, characterized in that that the DNA polymerase Taq polymerase is. Method according to claim 17, characterized in that that the DNA polymerase is ProHa DNA polymerase. Method according to claim 17, characterized in that that the DNA polymerase DNA polymerase beta from mammals. Process according to claims 1 to 15, characterized that the NTs * alpha phosphorothioate NTs * and the polymerase are the Klenow fragment of E. coli polymerase I or T4 DNA polymerase. A method according to claim 23, characterized in that the NT * is a nucleotide of the formula

is. A method according to claim 23, characterized in that the NT * is a nucleotide of the formula

is. A method according to claim 23, characterized in that the NT * is a nucleotide of the formula

is. Process according to claims 24 to 26, characterized that on the terminal amino group of the nucleotide is a fluorescent dye is attached. Process according to Claim 23, characterized in that the NT * is a nucleotide of the formula 7h) dNTP-SS-TRITC (L7)

is. Process according to Claim 23, characterized in that the NT * is a nucleotide of the formula 7i) dNTP-SS-Cy3 (L11)

is. Process according to claim 23, characterized in that the NT * is a nucleotide of the formula 7j) dNTP-SS-TRITC (L14)

is. Process according to claims 1 to 30, characterized that the Fluorescent dyes selected from the group consisting of cyanine dyes, Rhodamine, xanthene and their derivatives are selected. Kit to carry out the method according to the claims 1 to 31, characterized in that it has a reaction surface (a solid support), to carry out required reaction solutions, one or more polymerases, and nucleotides (NTs), of which one to four are labeled with fluorescent dyes, wherein the labeled NTs are further structurally modified (NT * or NTs *) that the Polymerase after incorporation of such NT * into a growing complementary strand unable to build another NT * in the same strand, wherein the fluorescent dye is cleavable and the structural Modification is a cleavable sterically demanding ligand. Kit according to claim 32, characterized in that the structural modification used bulky ligand the fluorescent dye used for labeling is. Kit according to claim 24 or 25, characterized that it further contains components: a) for the production of single strands from double strands required reagents, b) Nucleic acid molecules encoding PBS in the NSKFs introduced become, c) oligonucleotide primer, d) to split off the Fluorescent dyes and sterically demanding ligands required Reagents, and / or e) washing solutions. Kit according to claims 32 to 34, characterized that the reaction surface from the group consisting of silicone, glass, ceramics, plastics, Gels selected is. Kit according to claim 35, characterized in that the plastics Polycarbonates or polystyrenes are. Kit according to claim 35, characterized in that the gels Agarose or polyacrylamide gels. Kit according to claim 37, characterized in that the gels 1 to 2% agarose gels or 10 to 15% polyacrylamide gels. Kit according to claims 32 to 38, characterized that the DNA polymerase is a DNA polymerase without 3'-5 'endonuclease activity. Kit according to claim 39, characterized in that the polymerase from the group consisting of Sequenase-type viral DNA polymerases, beta-polymerases is selected from eukaryotes and thermostable DNA polymerases. Kit according to claim 40, characterized in that the DNA polymerase Sequenase Version 2, Polymerase Beta from Mammals, Taq polymerase or ProHa DNA polymerase. Kit according to claims 32 to 38, characterized that the NTs * alpha phosphorothioate NTs * and the polymerase are the Klenow fragment of E. coli polymerase I or T4 DNR polymerase is. Kit according to claims 32 to 42, characterized that the Fluorescent dyes selected from the group consisting of cyanine dyes, Rhodamine, xanthene and their derivatives are selected.