DE10214395A1 - Parallel sequencing of nucleic acid segments, useful for detecting single-nucleotide polymorphisms, by single-base extensions with labeled nucleotide - Google Patents

Abstract

Parallel sequence analysis of segments of a complete nucleic acid sequence, using sequence-specific primers, is new. Parallel sequence analysis of segments of a complete nucleic acid sequence, using sequence-specific primers comprises: (i) preparing single-stranded nucleic acid chain fragments (X) of 30-1000 nucleotides (nt), corresponding to overlapping parts of the complete sequence; (ii) immobilizing (X) on a flat surface; (iii) hybridizing one or more sequence-specific primer populations to (X), where the density of individual extension-capable (X)-primer complexes is 10-1000 micron-2;and (iv) performing a cyclic 'build-up' reaction of strands complementary to (X), comprising: (a) treating immobilized (X) with a solution of at least one polymerase, 1-4 nucleotides (nt) labeled with a fluorophore (and if more than one nt is used the fluorophores on each can be differentiated) and modified so that the polymerase can add only one nt to the growing complementary strand, and the group that causes termination can be removed, along with the fluorophore; (b) incubating to extend each complementary strand by just one nt; (c) washing; (d) detecting, from fluorescence, the nt incorporated into each strand, and simultaneously the relative position on the reaction surface; (e) cleaving the terminating group and fluorophore from the complementary strands; (f) washing; and (g) repeating steps (a)-(f), optionally many times. The relative positions of individual (X) on the surface and their sequence are determined from the signals detected during step (d). An Independent claim is also included for a surface for immobilizing (X) randomly at 10-106 per 100 micron-2.

Description

Summary

• a) Im Text wird allgemein unter einem "Primer" eine Population von Primermolekülen mit einheitlicher Struktur verstanden.
• b) "mehrere Primer" o. ä. werden im Text als mehrere Populationen von Primermolekülen verstanden, die unterschiedliche Struktur besitzen.
• c) Ein "Primer-Molekül" bedeutet ein einziges Oligonukleotid-Molekül.
• d) "Mehrere Primer-Moleküle" bedeuten mehrere einzelne Oligonukleotid-Moleküle, sie können einheitliche oder unterschiedliche Struktur aufweisen.

Primerbindungstelle (PBS) - Teil der Sequenz in der NSK oder NSKF, an den der Primer bindet.
Referenzsequenz - eine bereits bekannte Sequenz, zu der die Abweichungen in der zu untersuchenden Sequenz bzw. in den zu untersuchenden Sequenzen (Gesamtsequenz) ermittelt werden. Als Referenzsequenzen können in Datenbanken zugängliche Sequenzen verwendet werden, wie z. B. aus der NCBI-Datenbank.
RNA - Ribonukleinsäure
SMPS-Verfahren - Single-Molecule-Parallel-Sequencing-Verfahren mit Einsatz der Weitfeld-Optik-Detektionsvorrichtung.
SNP - Einzelnukleotidpolymorphismen (s. dort) SNP-Stelle - eine Position in NSK, die auf Vorhandensein oder Abwesenheit von SNP untersucht wird.
Tm - Schmelztemperatur
Weitfeld-Optik-Detektionsvorrichtung - Detektionsvorrichtung, die gleichzeitig Fluoreszenzsignale von einzelnen, auf einer Fläche von größer als 20 µm × 20 µm verteilten Molekülen detektieren kann. Beispiele für eine solche Apparatur sind in Anmeldung Tcherkassov et al. (Aktenzeichen 101 48 868.8 beim DPuMA) beim DMPA angegeben. Ein weiteres Beispiel für Weitfeld-Detektionsoptik stellt Fluoreszenzmikroskop Axiovert 200 oder Axioplan 2e (Zeiss) mit einem Planneofluar-Objektiv 100 × NA 1.4 Ölimmersion (Zeiss), oder einem Planapochromat-Objektiv 100 × NA 1.4 Ölimmersion (Zeiss); die Anregung der Fluoreszenz kann dabei mit einer Lampe, z. B. Quecksilberdampflampe, oder einem Laser erfolgen. Sowohl Epifluoreszenzmdus als auch im Totalreflexions-Fluoreszenzmikroskopie-Modus (total internal reflection fluorescence microscopy, TIRF-Microscopy) oder Laser-Scanning-Mikroskopie-Modus können verwendet werden.
Zielsequenz - Teil einer Gesamtsequenz, der durch die Verwendung eines spezifischen Primers in der Sequenzierungsreaktion sequenziert/bestimmt wird. Eine Gesamtsequenz kann mehrere Zielsequenzen enthalten. Eine Zielsequenz ist genügend lang, um eine Positionierung dieser Zielsequenz innerhalb der Gesamtsequenz mit großer Wahrscheinlichkeit zu gewährleisten. Zielsequenzen können beispielsweise eine oder mehrere SNP-Stellen enthalten. The invention relates to methods and a reaction surface for the analysis of nucleic acid chain sequences, in particular for the analysis of single nucleotide polymorphisms and mutations. These methods are based on the sequencing of individual nucleic acid chains fixed on the reaction surfaces according to the invention. The sequencing reaction takes place through the sequential construction of a strand complementary to individual single-stranded nucleic acid chains bound to the surface. The sequence for the bound nucleic acid chains is determined from the sequence of the incorporated nucleotides. 1. Abbreviations and explanations of terms DPuMA - German Patent and Trademark Office
DNA - deoxyribonucleic acid of different origins and different lengths: (genomic DNA, cDNA, ssDNA, dsDNA)
dNTP - 2'-deoxi-nucleoside triphosphates, substrates for the incorporation reaction of DNA polymerases and reverse transcriptases
Single nucleotide polymorphisms (SNPs) - are changes in the sequences that can occur as a substitution (transition or transversion) or as a deletion or insertion of individual NT.
Detection Sequence - Part of the target sequence that is used to assign this target sequence to the overall sequence.
Whole sequence - the sequence used in the sequencing reaction, mostly transferred to NSKFs. It can originally consist of one or more NSKs. The total sequence can represent parts or equivalents of another sequence or sequence populations (eg mRNA, cDNA, plasmid DNA with a cloned section, BAC, YAC) and can come from one or different species. In most cases, the overall sequence represents a population of many NSK molecules of the same structure.
NSK - nucleic acid chain. DNA or RNA in their original length
NSKF - nucleic acid chain fragment (DNA or RNA), which corresponds to a part of the total sequence, NSKFs - nucleic acid chain fragments. The sum of the NSKFs is equivalent to the overall sequence. The NSKFs can, for example, be fragments of the entire DNA or RNA sequence that arise after a fragmentation step.
NT - natural nucleotide, usually dNTP, unless expressly stated otherwise. Abbreviation "NT" is also used when specifying the length of a nucleic acid sequence, e.g. B. 1,000 NT. In this case, "NT" stands for nucleoside monophosphates. The majority of abbreviations in the text 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 stated otherwise. NTs * means: modified nucleotides
Flat surface - surface which preferably has the following features: 1) It allows several individual molecules, preferably more than 100, more preferably more than 1000, to be detected simultaneously with the given objective-surface distance at one objective position. 2) The immobilized or bound individual molecules are in the same focal plane, which can be set reproducibly.
Polymerases - enzymes that can incorporate complementary nucleotides into a growing strand of DNA or RNA (e.g. DNA polymerases, reverse transcriptases, RNA polymerases)
Primer - more specific, a sequence-specific primer oligonucleotide that binds complementarily to a specific, selected site in the overall sequence and serves as a primer for the sequencing reaction. To clarify the inventive concept, the following terms are distinguished in the text:

• a) In the text, a "primer" is generally understood to mean a population of primer molecules with a uniform structure.
• b) "Several primers" or the like are understood in the text as several populations of primer molecules which have different structures.
• c) A "primer molecule" means a single oligonucleotide molecule.
• d) "Several primer molecules" mean several individual oligonucleotide molecules, they can have a uniform or different structure.

Primer binding site (PBS) - part of the sequence in the NSK or NSKF to which the primer binds.
Reference sequence - a sequence already known, for which the deviations in the sequence to be examined or in the sequences to be examined (overall sequence) are determined. Sequences accessible in databases can be used as reference sequences, e.g. B. from the NCBI database.
RNA - ribonucleic acid
SMPS method - single-molecule parallel sequencing method using the wide-field optical detection device.
SNP - single nucleotide polymorphisms (see there) SNP site - a position in NSK that is examined for the presence or absence of SNP.
Tm - melting temperature
Wide-field optical detection device - detection device which can simultaneously detect fluorescence signals from individual molecules distributed over an area of more than 20 μm × 20 μm. Examples of such an apparatus are in the application Tcherkassov et al. (File number 101 48 868.8 at the DPuMA) stated at the DMPA. Another example of wide-field detection optics is the Axiovert 200 or Axioplan 2e (Zeiss) fluorescence microscope with a Planeofluar objective 100 × NA 1.4 oil immersion (Zeiss), or a planapochromatic objective 100 × NA 1.4 oil immersion (Zeiss); the excitation of fluorescence can be done with a lamp, e.g. B. mercury vapor lamp, or a laser. Both epifluorescence mode and total internal reflection fluorescence microscopy (TIRF microscopy) mode or laser scanning microscopy mode can be used.
Target Sequence - Part of an overall sequence that is sequenced / determined using a specific primer in the sequencing reaction. An entire sequence can contain several target sequences. A target sequence is long enough to ensure that it is very likely to be positioned within the overall sequence. Target sequences can contain one or more SNP sites, for example.

2. State of the art

Nucleic acid chain sequence analysis is in many areas of science, medicine and industry has become an important tool. Several were used for analysis Process developed.

The best known methods are chain termination sequencing according to Sanger (F. Sanger et al. PNAS 1977 v. 74 s. 5463), which is based on the installation of chain terminators, and the Maxam-Gilbert method, which is based on base-specific modification and cleavage of nucleic acid chains (A.M. Maxam and W. Gilbert PNAS 1977, v. 74 p. 560). Both methods deliver a number of different nucleic acid chain fragments Lengths. These fragments are separated lengthwise in a gel. Doing so all disadvantages of electrophoresis (such as long running times, relatively short distances from Sequences that can be determined in one approach, limited number of parallel ones Approaches and relatively large amounts of DNA) are accepted. These methods are very labor intensive and slow.

Another method for sequencing is based on the hybridization of Nucleic acid chains with short oligonucleotides. This is done with mathematical Methods calculates how many oligonucleotides of a certain length are present to determine a complete sequence (Z. T. Strezoska et al. PNAS 1991 v. 88 S. 10089, R. S. Drmanac et al. Science 1993 v. 260 p. 1649). This procedure is also included Problems: Only one sequence can be determined in one approach.

WO 00/56925 describes a method which can detect many SNPs in parallel. One of the biggest disadvantages of this and similar process is the amount of material that you have required for the identification of SNPs. Another disadvantage is the need to fix certain primers to certain particles or positions on a surface (spatial or other coding of oligonucleotides) to make statements about SNPs to be able to. Another disadvantage is the great cost involved.

In registration Tcherkassov et al. (File number 101 20 797.2-41 DPuMA) were SBS- Process (Sequencing by Synthesis) for the parallel sequencing of individual Nucleic acid chain molecules described.

The invention represents a further development of the SBS method with single molecules.

The first object of the invention is a method which involves many relevant single nucleotide Polymorphisms (SNPs) identified quickly and simultaneously, not a predetermined one Coding of oligonucleotides (e.g. by a defined positioning of Oligonucleotides on a surface or a defined color coding of beads with defined oligonucleotides) and requires little material.

Another object of the invention relates to a method for analyzing Sequence sections (target sequences) of an overall sequence with sequence-specific Primers.

Another object of the invention is a surface with immobilized Nucleic acid chains, NSKFs.

3. General description

The invention relates to methods and reaction surface for the analysis of NSC sequences, in particular for analyzing sequence sections of an overall sequence Single nucleotide polymorphisms and mutations.

This method is based on the sequencing of individual, on the reaction surface fixed NSK or NSKF molecules (SMPS method). During the Sequencing reactions are labeled NT * s in the growing strand by a Polymerase built in and the signals from individual built-in NT * molecules detects Tcherkassov et al. Case number 101 20 797.2-41 DPuMA. After installation and upon detection of the signal, the signal is removed and a next complementary NT * incorporated by a polymerase. From the order of the inserted nucleotides the sequence for the bound nucleic acid chains is determined.

A procedure for the analysis of SNPs is used for clarity, not for limitation described in detail.

In one embodiment of the inventive method for SNP analysis several potential SNP positions in the reference sequence are selected, which in one too analyzing NSC are examined. These positions are accordingly different, sequence-specific primers are provided. These primers can do one form standardized primer set for SNP analysis for a specific question and used consistently as a kit for the analyzes in question.

The preparation of the material to be analyzed (individual and double-stranded nucleic acid chains) depends on the task and has according to the invention, the goal of one or more long nucleic acid chains (Total sequence) a population of relatively small, between 30 and 2000 NT long, to form single-stranded nucleic acid chain fragments (NSKFs).

These NSKF molecules are randomly immobilized on a flat surface with a density between 10 and 1,000,000 per 100 µm ² . Primers are hybridized to the NSKFs bound on the surface, so that the density of the NSKF-primer complexes capable of extension is approximately 10-1000 per 100 µm ² .

After the hybridization, unbound primers are removed and the Sequencing reaction started. The synthesis of the complementary strand to each bound NSKF molecule. In doing so Marked NTs * built into the newly synthesized strand. The polymerase just builds in only marked NT * in the growing chain. This can be done, for example, by reversible coupling of a group leading to termination to the NTs * achieved become. The installation of another marked NT * is initially impossible made.

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

The sequencing reaction preferably proceeds in several cycles. A cycle includes the following steps, for example:

• a) adding a solution with labeled nucleotides (NTs *) and polymerase to the NSKF primer complexes,
• b) incubation of the NSKF primer complexes with this solution under conditions which are suitable for extending the complementary strands by one NT,
• c) washing,
• d) detection of the signals of the individual built-in NT * molecules,
• e) removal of the marking and the group leading to the termination from the installed NT *,
• f) washing.

If necessary, the cycle is repeated several times (a-f).

The reaction conditions of step (b) in one cycle are chosen so that the Polymerases on more than 50% of the NSKF- involved in the sequencing reaction Primer complexes can incorporate a marked NT * in one cycle, preferably on more than 95%.

The number of cycles to be carried out depends on the task at hand, is theoretically not restricted and is between 2 and 5000, preferably between 4 and 500.

• 1. Durch eine große Parallelität der Sequenzierung (theoretisch unbegrenzte Anzahl, praktisch bis zu 10.000.000 der zu analysierenden NSKFs) kann man viele SNPs, bzw. Zielsequenzen gleichzeitig analysieren. Die Redundanz der Daten erlaubt die Verifikation der Sequenzen und die Korrektur von Fehlbestimmungen sowie die Ermittlung des homo- und heterozygoten Zustandes der SNPs im jeweiligen Träger.
• 2. Es ist nicht notwendig, NSKF-Moleküle in einer definierten Anordnung auf der Oberfläche zu fixieren, da das Signal von einzelnen Molekülen ausgeht und nicht von einer räumlich definierten Population von Primern.
• 3. Durch eine Auswahl der Zielsequenzen und der sequenzspezifischen Primer werden nur die relevanten Abschnitte der Gesamtsequenz untersucht, was die Menge nicht relevanter Informationen verringert und die Analysezeit verkürzt.
• 4. Durch eine hohe Konzentrationen der sequenzspezifischen Primer in der Hybridisierungslösung wird die Hybridisierungszeit der Primer an die NSKFs deutlich verkürzt.
• 5. Die Zahl der Vorbereitungsschritte des Materials für die Sequenz-Analyse ist minimal.

The process according to the invention and the surface result in several advantages:

• 1. Due to the great parallelism of the sequencing (theoretically unlimited number, practically up to 10,000,000 of the NSKFs to be analyzed), many SNPs or target sequences can be analyzed simultaneously. The redundancy of the data allows the verification of the sequences and the correction of incorrect determinations as well as the determination of the homo- and heterozygous state of the SNPs in the respective carrier.
• 2. It is not necessary to fix NSKF molecules in a defined arrangement on the surface, since the signal comes from individual molecules and not from a spatially defined population of primers.
• 3. By selecting the target sequences and the sequence-specific primers, only the relevant sections of the overall sequence are examined, which reduces the amount of irrelevant information and shortens the analysis time.
• 4. A high concentration of the sequence-specific primers in the hybridization solution significantly shortens the hybridization time of the primers to the NSKFs.
• 5. The number of preparation steps of the material for the sequence analysis is minimal.

The following registrations are fully cited here:
Patent application Tcherkassov et al. Case number 101 49 786.5 at the DPuMA
Patent application Tcherkassov et al. Case number 101 48 868.8 at the DPuMA
Patent application Tcherkassov et al. File number 101 20 797.2-41 at the DPuMA
Patent application Tcherkassov et al. File number 101 20 798.0-41 at the DPuMA
Patent application Tcherkassov et al. Case number 101 42 256.3 at the DPuMA

4. Detailed description of the invention

• 1. Zur Analyse jeder ausgewählten SNP-Stelle werden spezifische Primer bereitgestellt, so dass jede zu untersuchende SNP-Stelle entweder die nächste Position 3'-wärts vom Primer einnimmt oder innerhalb von 2 bis 100, vorzugsweise 2 bis 50, idealerweise 2 bis 20 Positionen 3'-wärts vom Primer liegt. Die SNP- Stelle liegt somit innerhalb der Zielsequenz, die während der Sequenzierungsreaktion bestimmt wird. Es werden vorzugsweise mehrere SNP- Stellen gleichzeitig analysiert, so dass mehrere spezifische Primer verwendet werden müssen. Die Primer werden vorzugsweise so ausgewählt, dass sie möglichst einheitliche Annealing-Temperaturen haben.
• 2. Von der Gesamtsequenz werden kurze Nukleinsäurekettenfragmente (NSKFs) abgeleitet, wobei diese Fragmente einzelsträngig sind und eine Länge von 20 bis 2000 NT, vorzugsweise 30 bis 500 NT besitzen.
• 3. NSKF-Moleküle werden in einer zufälligen Anordnung auf der Oberfläche immobilisiert.
• 4. Nach der Hybridisierung (Annealing) von sequenzspezifischen Primern an die auf der Oberfläche immobilisierten NSKFs wird eine zyklische Sequenzierungsreaktion durchgeführt, wobei für jedes an der Reaktion beteiligte NSKF-Molekül eine Zielsequenz ermittelt wird. Die Sequenzierungsreaktion läuft an vielen Molekülen gleichzeitig ab.
• 5. Die ermittelten Zielsequezen enthalten Information über die Zugehörigkeit zu einem bestimmten Abschnitt in der Gesamtsequenz und über den SNP in diesem Abschnitt bei der zu untersuchenden Probe. Die Länge der Zielsequenzen und somit die Zahl der Zyklen ist so zu wählen, dass eine Identifizierung der Sequenzen gewährleistet werden kann.

The method according to the invention for SNP analysis is based on the following principles:
Locations in a reference sequence are selected which are to be checked for single nucleotide polymorphisms (SNPs) in the NSKs to be examined (overall sequence)

• 1. Specific primers are provided for the analysis of each selected SNP site, so that each SNP site to be examined either occupies the next position 3 'to the primer or within 2 to 100, preferably 2 to 50, ideally 2 to 20 positions 3'-is from the primer. The SNP site is thus within the target sequence that is determined during the sequencing reaction. Several SNP sites are preferably analyzed simultaneously, so that several specific primers have to be used. The primers are preferably selected so that they have annealing temperatures that are as uniform as possible.
• 2. Short nucleic acid chain fragments (NSKFs) are derived from the total sequence, these fragments being single-stranded and having a length of 20 to 2000 NT, preferably 30 to 500 NT.
• 3. NSKF molecules are immobilized in a random arrangement on the surface.
• 4. After the hybridization (annealing) of sequence-specific primers to the NSKFs immobilized on the surface, a cyclic sequencing reaction is carried out, a target sequence being determined for each NSKF molecule involved in the reaction. The sequencing reaction takes place on many molecules simultaneously.
• 5. The determined target sequences contain information about the belonging to a specific section in the overall sequence and about the SNP in this section for the sample to be examined. The length of the target sequences and thus the number of cycles should be chosen so that an identification of the sequences can be guaranteed.

In an advantageous embodiment, the determined target sequences are compared with the reference sequence and assigned by sequence matching. If the target sequence is sufficiently long, it is very likely that it can be assigned to a specific position in the reference sequence. For example, a sequence of 10 NTs can form more than 10 ⁶ different combinations and can therefore be identified with a high probability in a NSK of only 100,000 NT. After assigning the determined target sequence to the specific position within the reference sequence, differences in the sequences, the SNPs, become visible.

Another is advantageous for identifying the target sequences Embodiment both the already known number of primers, their Composition and an already known, to the primer binding site subsequent sequence section of the reference sequence used. The determined target sequences analyzed according to their affiliation to the primers, only the sequences close to the primer binding site are taken into account Need to become. For example, if only 1000 primers are used, it is sufficient less than 10 NTs of the determined target sequences in order to be assigned to the to enable appropriate primers.

material selection

With the aid of the method according to the invention, it is possible to use both preselected DNA Sequences (e.g., sections of a genome cloned into YAC, PAC, or BAC vectors (R. Anand et al. NAR 1989 v. 17 p. 3425, H. Shizuya et al. PNAS 1992 v. 89 p. 8794, "Construction of bacterial artificial chromosome libraries using the modified PAC system" in "Current Protocols in Human genetics" 1996 John Wiley & Sons Inc)) as well preselected DNA (e.g. genomic DNA, cDNA or mRNA mixtures or their Equivalents). Through careful primer selection, it is possible in one search for a large amount of genetic information specifically for specific SNP positions.

The sample to be analyzed usually contains several identical total sequence molecules, z. B. multiple copies of genomic DNA from cells of a tissue or multiple identical mRNA populations from cells of a tissue.

material preparation

The aim of the material preparation is to produce single-strand NSKFs with a length between 30 and 2000 NTs, preferably 30-500 NTs and a single primer binding site receive. To improve clarity, here are some examples, the mentioned methods can be used individually or in combination.

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

The fragmentation is necessary so that only one primer per NSKF can bind. According to the invention, the nucleic acid chain fragments (NSKFs) can be generated by several methods are used, e.g. B. with the fragmentation of the starting material Ultrasound or by endonucleases ("Molecular cloning" 1989 J. Sambrook et al. Cold Spring Harbor Laborotary Press), e.g. B. by unspecific endonuclease mixtures. Ultrasonic fragmentation is preferred. You can set the conditions that fragments with an average length of a few hundred NTs arise.

You can also use one long NSK or multiple long NSKs randomized primer complementary even shorter NSKFs can be synthesized. This method is particularly preferred for the analysis of SNPs in mRNA or cDNA populations. Single-stranded DNA fragments are included in the mRNA randomized primers and a reverse transcriptase (Zhang-J et al. Biochem. J. 1999 v. 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. 27 p. 962).

Single-stranded preparation

Single-stranded NSKFs are required for the sequencing reaction. If that Starting material is in double-stranded form, there are several ways out double stranded DNA to produce a single stranded form (e.g. heat denaturation or alkali denaturation) ("Molecular cloning" 1989 J. Sambrook et al. Cold Spring Harbor Laborotary Press).

SNP sites choice

With the method according to the invention, both known SNP points can be analyzed as well as identify new SNP positions. Any position in the NSK occur. The selection depends on the question, e.g. B. SNP analysis in Genes whose products are associated with certain diseases, or SNP analysis in conserved, coding sections of the genes that code for membrane receptors, or checking known SNP sites in regulatory sequences of genes that are responsible for cell division is important.

An SNP site to be analyzed lies within a target sequence that occurs during the Sequencing reaction is determined. You can have multiple SNP positions within one Determine target sequence. On the other hand, you can also target multiple sequences. B. within choose a gene. It is important that the target sequences are at a sufficient distance from each other in the overall sequence. This distance is necessary so that only one sequence specific primer hybridized per NSKF, and it is of average NSKF length dependent: the shorter the NSKFs, the closer to each other can be Target sequences lie. With an appropriate choice of primers, the SNP sites can do both Strands of a double-stranded nucleic acid chain are analyzed.

The procedure also offers the option of, for example, several SNP positions from many To control individuals (as a sample of a population) at the same time. This can z. B. the SNP profile of a population can be examined.

Primer for the sequencing reaction

Sequencing reaction on a single NSKF molecule is by a primer Molecule enables. According to the invention, a sequence-specific primer is necessary in order to the sequencing reaction in each case on a specific / specific target sequence to be able to perform within the overall sequence. The one for the analysis of an SNP Site, or a target sequence to be used sequence-specific primer provides Population of primer molecules with identical structure. For the analysis of several, different target sequences are several different primer populations necessary.

By using sequence-specific primers, only the relevant ones Sequence sections, the target sequences, are analyzed. In the method according to the invention the length of the sequences to be sequenced is kept as short as possible so that the Analysis speed increases.

• 1. Die zu analysierende SNP-Stelle sollte entweder gleich nach dem 3'-Ende des Primers oder innerhalb der nächsten 2 bis 50 NTs, vorzugsweise 2 bis 20 NTs liegen.
• 2. Die Positionierung (die Wahl der Sequenzlänge und der Zusammensetzung) der PBS zu SNP-Stelle sollte so erfolgen, dass die verschiedenen PBS-Sequenzen und die korrespondierenden Primer-Sequenzen möglichst ähnliche "Annealing-temperaturen" besitzen, um bei möglichst einheitlichen Hybridisierungsbedingungen zu binden. Das kann beispielsweise durch Veränderung der PBS-Position im Bezug auf die jeweilige, zu analysierende SNP-Stelle oder durch die Veränderung der Primersequenzlänge erfolgen (Rychlik et al. NAR 1990 v. 18 S. 6409).
• 3. Der minimale Abstand zwischen Primern, die an denselben Strang in der Gesamtsequenz binden, sollte die durchschnittliche NSKF-Länge nicht unterschreiten.

A sequence-specific primer binds to a specific primer binding site in the sequence to be analyzed, PBS. The composition and length of the primers are optimized for each potential SNP site or target sequence. Examples of optimization steps are in Rychlik et al. NAR 1990 v. 18 p. 6409. When choosing the primer or choosing the PBS (primer binding site), the following aspects must be considered:

• 1. The SNP site to be analyzed should either be immediately after the 3 'end of the primer or within the next 2 to 50 NTs, preferably 2 to 20 NTs.
• 2. The positioning (the choice of the sequence length and the composition) of the PBS at the SNP site should be carried out in such a way that the different PBS sequences and the corresponding primer sequences have “annealing temperatures” that are as similar as possible in order to achieve the most uniform hybridization conditions possible tie. This can be done, for example, by changing the PBS position in relation to the respective SNP site to be analyzed or by changing the length of the primer sequence (Rychlik et al. NAR 1990 v. 18 p. 6409).
• 3. The minimum distance between primers that bind to the same strand in the overall sequence should not be less than the average NSKF length.

Primers can be used for both strands of a double-strand NSK. In order to For example, you can record SNP points that are close to each other, or you can carry out a check of an SNP point in both lines.

The length of the primer is preferably between 6 and 100 NTs, optimally between 10-30 or 30-40 or 40-50. For different SNP positions or target sequences primers with different lengths can be used.

The synthesis of such a primer can e.g. B. with the DNA synthesizer 380 A from Applied Biosystems can be executed or as a custom synthesis at a commercial providers, e.g. B. MWG-Biotech GmbH, Germany.

For the SNP analysis with sequence-specific primers, primers are used according to the invention in a hybridization solution to the NSKFs immobilized on the reaction surface hybridizes (annealing reaction).

Immobilization of NSKFs

The NSKF molecules provided are randomized to the flat surface Arrangement bound.

The surface of a solid phase of a material serves as a flat surface is preferably inert towards enzymatic reactions and no interference with the Detection causes. Silicon, glass, ceramics, plastics (e.g. polycarbonates or Polystyrene) or any other material that meets these functional requirements is sufficient, can be used. The surface is preferably not deformable because otherwise the signals are likely to be distorted during repeated detection.

If a gel-like solid phase is used, this gel can e.g. B. an agarose or polyacrylamide gel. The gel is preferably freely passable for molecules with a molecular mass below 5000 Da (for example, a 1 to 2% agarose gel or 10 to 15% polyacrylamide gel can be used). Such a gel surface has the advantage over other solid surfaces that the marked NSKFs, in contrast to free NTs *, are held on the surface. By fixing the NSKFs on the surface, it is possible to detect the fluorescence signals from built-in NTs *. The signals from free NTs * are not detected because they do not bind to the material of the gel and are therefore not immobilized. The gel is preferably attached to a solid surface. The thickness of the gel is preferably not more than 0.1 mm. However, the gel thickness must be greater than the depth of field of the lens, so that NTs * bound non-specifically to the solid base do not reach the focal plane and are therefore detected. If the depth of field e.g. B. 0.3 µm, the gel thickness should preferably be between 1 µm and 100 µm. The reaction surface must be large enough to be able to fix the necessary number of NSKFs with the appropriate density. The reaction surface should preferably not be larger than 20 cm ² . A preferred surface, a gel surface, is detailed in the application Tcherkassov et al. Case number 101 49 786.5 described at DPuMA.

The NSKFs are preferably immobilized at one of the two chain ends. This can be achieved by appropriate covalent (eg Tcherkassov et al. (File number 101 49 786.5 at DPuMA), affine (eg biotin-streptavidin) or other bonds. Many examples of immobilization of nucleic acids are known (McGall et al. US Patent 5412087, Nikiforov et al. US Patent 5610287, Barrett et al. US Patent 5482867, Mirzabekov et al. US Patent 5981734, "Microarray biochip technology" 2000 M. Schena Eaton Publishing, "DNA Microarrays" 1999 M. Schena Oxford University Press, Rasmussen et al. Analytical Biochemistry v. 198, p. 138, Allemand et al. Biophysical Journal 1997, v. 73, p. 2064, Trabesinger et al. Analytical Chemistry 1999, v. 71, p. 279 , Osborne et al. Analytical Chemistry 2000, v. 72, p. 3678, Timofeev et al. Nucleic Acid Research (NAR) 1996, v. 24 p. 3142, Ghosh et. Al. NAR 1987 v. 15 p. 5353, Gingeras et al. NAR 1987 v. 15 p. 5373, Maskos et al. NAR 1992 v. 20 p. 1679. The immobilization can also be carried out by an unspecific binding, e.g. , B. the drying of the NSKFs containing sample can be achieved on the flat surface. The density of the immobilization can be between 10 and 1,000,000 NSKFs per 100 µm ² .

The different cycle steps require an exchange of the different ones Reaction solutions above the surface. The reaction surface is preferred Part of a reaction vessel. The reaction vessel is again preferred Part of a reaction apparatus with a flow device. The flow device enables the solutions in the reaction vessel to be exchanged. The exchange can be with a computer-controlled pumping device or manually. Important it is that the surface does not dry out. The volume of the Reaction vessel less than 50 µl. Ideally, its volume is less than 1 µl. An example of a flow device is described in the patent application Tcherkassov et al. Case number 101 48 868.8 described at the DPMA.

hybridization

The immobilized NSKFs and the primers are under stringent Hybridization conditions are incubated, which is as selective as possible Allow (annealing) of the primers to the corresponding primer binding sites of the NSKFs. Optimal hybridization conditions depend on the exact structure of the Primer binding sites and the respective primer structures and can for example according to Rychlik et al. NAR 1990 v. 18 p. 6409 calculate.

The primers are preferably a mixture of primers. The concentrations of individual sequence-specific primers (individual concentrations of primer populations) preferably between 10 pmol / l and 100 µmol / l. The total concentration of primers in the The primer mixture is preferably between 1 nmol / l and 10 mmol / l. The relationship between individual primer populations can vary. Primers can be clearly Excess over the immobilized NSKFs are added so that the Hybridization time is short.

The density of NSKF primer complexes that can be extended for the SMPS is approx. 10 to 1000 per 100 µm ² . It can be achieved before, during or after hybridization of the primers.

In the case of a known NSKF concentration, in one embodiment the immobilization conditions can be selected such that the NSKFs are bound in a density of approx. 10 to 1000 molecules per 100 µm ² . NSKFs thus determine the density of the NSKF primer complexes.

In another embodiment, the density of the immobilized NSKFs can be substantially higher than 1000 NSKFs per 100 µm ² , e.g. B. 1000,000 per 100 microns ² . The density of the NSKF primer complexes required for optical detection is achieved during the primer hybridization. The hybridization conditions (e.g. temperature, time, buffer) should be selected so that the primers only bind to a part of the immobilized NSKFs.

If the NSKF concentration is unknown and accordingly unknown Immobilization density can lead to the hybridization (annealing) of primers to the NSKFs lead to a higher than optimal density of NSKF primer complexes.

For this reason, in an advantageous embodiment, part of the NSKFs containing sample used for determining the optimal density. This part is on immobilized on a reaction surface, the primers are hybridized to the NSKFs and the resulting NSKF primer complexes are built in by Fluorescent dye-bearing NT * s (e.g. Cy3-dCTP, Amersham Pharmacia Biotech) marked. On the one hand, the density that is determined can be used to determine any necessary Dilution or concentration of the original sample for the final one Calculate the sequencing approach (the hybridization conditions are maintained). On the other hand, necessary changes in the Hybridization conditions are calculated, for example a shortening of the Hybridization time, with the NSKF immobilization density remaining constant.

Another possibility to set the optimal density of extensions NSKF primer complexes consist in the controlled removal of excess NACF primer complexes.

After the hybridization of primers to the fixed NSKFs, a Labeling reaction performed in which all NSKF primer complexes capable of extension be marked. The primers should have the same melting temperature (Tm) as possible. An example of such a marking is the installation of reversible terminators (Tcherkassov et al file number 101 20 797.2-41 at the DPuMA). After The surface is washed and the NT * not installed is removed. The Initial density of the NSKF primer complexes is determined using the detection apparatus analyzed and if it is too high, it is reduced in a controlled manner.

The temperature of the solution above the surface becomes continuous or gradual increases and at the same time the density of the signals from in the NSKF primer complexes built-in NT * s checked. Both can be done, for example, in an automatic sequencer according to Tcherkassov et al file number 101 48 868.8 at the DPuMA. With increasing Temperature disintegrate the NSKF primer complexes, so that the primers are built in Transfer NT * s to the solution above the surface. After reaching the optimum density, the temperature is lowered and the marking is removed.

All 4 NT * s are preferably provided with a uniform marking. The The solution temperature can be checked differently, for example by means of a direct heating of the reaction vessel, including the reaction surface, or by a direct supply of the solution, which is heated outside the reaction vessel.

The quantity ratio between primer populations can be different or the same be great. With a higher primer concentration, certain, for example rarer sequences with a higher probability in a certain period of time be bound.

The great advantage of the method arrangement according to the invention over one Process arrangement with sequence-specific immobilized on a surface Primers and a subsequent hybridization of samples to these primers is the significant reduction in the time for hybridization between the sequence-specific Primers and the samples to be analyzed on the reaction surface.

detection

Example of the principles of detection of signals from built-in NT * and more required equipment are in the patent applications at DPuMA Tcherkassov et al. Case number 101 48 868.8.

Specification of the detection conditions

The incorporation of the labeled nucleotides is preferably carried out using optical means such. B. Wide field fluorescence microscope with an epifluorescence or a total internal Reflection lighting (total internal reflection excitation), or a laser scanning Microscope ("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 2nd ed. Herman BIOS Scientific Publishers, "Handbook of biological confocal microscopy" 1995 J. Pawley Plenum Press), an automatic sequencer (Tcherkassov et al. file number 101 48 868.8 at DPuMA) or a similar optical detection device with wide field optics and one Resolution of approx. 200-300 nm was detected. The use of an automatic sequencer or of a device with similar optical properties is compared to the use of Near-field microscopy or other high-resolution technology (with a resolution of a few nanometers) is particularly advantageous because it has a much larger surface area (preferably larger than 20 µm × 20 µm) with a large number of relevant signals in a short time can be analyzed. Use is made in the present invention made by this wide field optics detection technique.

incorporation reaction

Examples of a cyclic sequencing reaction, the NT * structures and the Polymerases are described in detail in the patent applications Tcherkassov et al. File number 101 20 797.2-41, 101 20 798.0-41, 101 42 256.3 at DPuMA. It can four labeled NT * s are used in the reaction as well as two labeled NT * s and two unmarked NT.

5. Examples Gel Preparation

The preparation of the surface is described in the registration Tcherkassov et al. (File number 101 49 786.5 at DPuMA) described in detail.

The polyacrylamide gel for the analysis of reactions with single molecules is used General rules of gel preparation for electrophoretic separation are created ("Electrophoresis" A.T. Andrews, Oxford science publications 1995).

The polymerization reaction can e.g. B. be carried out by UV light or by radical formers. In this example, ammonium persulfate (APS) and TEMED (tetramethylethylenediamine) are used for the radical reaction, e.g. B. TEMED 0.01% v / v and APS 0.04% w / v. The component composition can vary widely, the concentrations of individual components are in the following ranges (calculated for the ready-to-use aqueous AA-bisAA solution):
Acrylamide monomer (AA) from 3 to 30%, ideally between 10 and 20%
Bis-acrylamide (bis-AA) in relation to the acrylamide monomer 1:10 to 1:50, preferably 1:20.

Two clean glass plates are used for production (with acetone and then water washed). A glass plate (P1) is preferably made with a water-repellent reagent pretreated, e.g. B. Repelsilane, dimethyldichlorosilane solution, Amersham Pharmacia- Biotech. P2 serves as a solid carrier for the gel and can be used with yellow-binding reagents e.g. B. Bind silane, methacryloxypropyltrimethoxysilane, Amersham Pharmacia-Biotech, be pretreated so that there is a covalent bond between the gel and the Glass surface is coming. The P2 pretreatment with yellowing reagents is then useful if several reactions with immobilized molecules are carried out have to. With a smaller number of reactions, such pretreatment is not necessary. In these cases, a clean glass surface is sufficient for P2 so that the gel adheres to the glass surface solely through adhesive forces.

The finished polymerization solution (AA / bisAA solution with radical formers) is between P1 and P2 cast so that a layer of about 5 to 30 microns results. The thickness of the gel can e.g. B. be controlled by spacers. After hardening, P1 is removed. The Gel sticks to P2. It is washed with deionized water.

The gel can be used directly or at different stages of manufacture be dried and stored. Before reacting with labeled molecules, this will Gel usually swelled in the reaction buffer solution for a few minutes and first then used for the reaction.

NSKFs are dried out on a gel surface prepared in this way immobilized.

For example, a solution (approx. 1 μl) of a plasmid DNA (pMOS blue plasmid DNA linearized with Hind III, converted into single-stranded form by heat) was approx. 3400 NT long, concentration 0.1 μg / μl) to approx. 10 mm Dripped ^{2 of} the gel surface and brought to dry at 90 ° C. The calculated density of the immobilized plasmid molecules was approximately 1000 per 1 µm ² .

The oligonucleotide 5'-AGTGAATTCGAGCTCGGTAC-3 'was used as primer. The primer binding site (hereinafter bold) with the extension relevant for the analysis has the following sequence:

A flow cell (microfluidic channel, MFK according to Tcherkassov et al. File number 101 48 868.8 with the DPuMA) was assembled with the reaction surface. Such a MFK allows a quick exchange of liquid over the gel surface.

As a preliminary test, the primer (calculated Tm 45.3 ° C, 0.1 µmol / l in 50 mmol / l Tris-HCl pH 8.7) was hybridized at 45 ° C for 10 minutes with the plasmid DNA on the surface (annealing) , After a washing step, the density of the plasmid-primer complexes was checked. The control was carried out by incorporating dCTP-Cy3 (Amersham Pharmacia Biotech) using Klenow fragment (2 units per 50 μl in 20 mmol / l Tris-HCl buffer, pH 8.5, with 5 mmol / l MgCl ₂ , 15 minutes at 30 ° C). Only a single dCMP-Cy3 is built into the growing strand.

Axioplan 2e (Zeiss) with the AxioCam CCD camera served as the detection apparatus (Zeiss). The detection apparatus and the evaluation of the signals are in the registration (Tcherkassov et al file number 101 20 797.2-41 at the DPuMA).

The signal density of the individual built-in dCMP-Cy3 molecules corresponds to the density of the plasmid-primer complexes which can be extended. Under the conditions mentioned, the density of the plasmid-primer complexes averaged approximately 15 per 100 μm ² and was thus of the desired order.

A cyclic sequencing reaction is carried out on a second surface prepared in the same way (pMOS blue plasmid DNA linearized with Hind III and converted into single-stranded form by heat for about 3400 NT, concentration 0.1 μg / μl with hybridized primers). Here, dUTP-SS-R-Cy3 ( Fig. 1) and dCTP-SS-R-Cy3 ( Fig. 2) are used as reversible terminators (Tcherkassov et al file number 101 20 797.2-41 at DPuMA). The detection apparatus is the same as in the preliminary test.

• a) Reaktionslösung für die Einbaureaktion: 20 mmol/l Tris-HCl-Puffer, pH 8.5, 5 mmol/l MgCl₂, 10% Glycerin, Klenow-Fragment (Amersham Pharmacia-Biotech) 2 U pro 50 µl (dialysiert gegen 20 mmol/l Tris-HCl, pH 8.5), dUTP-SS-R-Cy3 bzw. dCTP-SS-R-Cy3, oder dATP und dGTP je 10 µmol/l.
• b) Waschlösung: 20 mmol/l Tris-HCl pH 8.5, 0.01% Na-Azid
• c) Reaktionslösung für die Abspaltungsreaktion: 20 mmol/l Tris-HCl, pH 8.5, 50 mmol/l Merkaptoethanol.

The solutions used for the cyclic sequencing reaction are composed as follows:

• a) Reaction solution for the installation reaction: 20 mmol / l Tris-HCl buffer, pH 8.5, 5 mmol / l MgCl ₂ , 10% glycerol, Klenow fragment (Amersham Pharmacia-Biotech) 2 U per 50 µl (dialyzed against 20 mmol / l Tris-HCl, pH 8.5), dUTP-SS-R-Cy3 or dCTP-SS-R-Cy3, or dATP and dGTP each 10 µmol / l.
• b) Wash solution: 20 mmol / l Tris-HCl pH 8.5, 0.01% Na azide
• c) Reaction solution for the cleavage reaction: 20 mmol / l Tris-HCl, pH 8.5, 50 mmol / l mercaptoethanol.

The installation reactions with marked NT * s were carried out at 30 ° C. for 15 minutes. In the first cycle of the sequencing reaction, a reaction solution with dCTP-SS-R- Cy3 added. After a washing step, a detection step was carried out, whereby Single molecule signals with the assigned x, y coordinates registered on the surface (approximately 11,200 signals). Then the marking was removed by the built-in NT * s split off (room temperature, 10 minutes) and the surface washed.

In the second cycle, a reaction solution with dUTP-SS-R-Cy3 was added and 15 Incubated for minutes at 30 ° C. After a subsequent washing step, the Single molecule signals detected on the surface (approx. 200 signals). This matches with the background signal, which is caused by an unspecific binding of the NT * s to the surface arises. The marker from the NT * s was split off (room temperature, 10 Minutes) and the surface washed with the washing solution.

In the third cycle, a reaction solution with dATP and dGTP was added and 15 Incubated for minutes at 30 ° C. The surface was then washed.

Cycles 1 to 3 were repeated three times, with a total of approx. 9900 CCU Target sequences were determined. These target sequences can clearly become a primer be assigned.

Legend for Fig. 1 and 2

Nucleotide analogs are shown that lead to reversible termination. It works the dye (Cy3) coupled to the base via a linker as one for termination leading group (Tcherkassov et al file number 101 20 797.2-41 at the DPuMA). To the installation of the NT * in the complementary strand and the detection of the signals removed this group by cleaving the disulfide bond so that another NT * can be installed.

Claims (10)

1. A method for parallel sequence analysis of sequence sections of an entire sequence with sequence-specific primers, which includes the following steps: 1.1.1. Provision of single-stranded NSKFs with a length of approximately 30 to 1000 nucleotides, which correspond to overlapping partial sequences of the overall sequence 2. 1.2. Immobilization of NSKF molecules on a flat surface in a random arrangement 3. 1.3. Hybridization (annealing) of one or more sequence-specific primer populations to the immobilized NSKFs, the density of the individual NSKF-primer complexes capable of extension being between 10 and 1000 per 100 μm ² . 4. 1.4. Carrying out a cyclical build-up reaction of the strands complementary to NSKFs, which includes the following steps: a) Adding a solution to the bound NSKFs which contains one or more polymerases and one to four modified nucleotides (NTs *) which are labeled with fluorescent dyes, the fluorescent dyes present on the NTs * in each case with the simultaneous use of at least two NTs * are chosen so that the NTs * used can be distinguished from one another by measuring different fluorescence signals, and the NTs * are structurally modified such that the polymerase is not able to insert another NT after incorporating such an NT * into a growing complementary strand * to be built into the same strand, whereby the group leading to the termination can be split off with the fluorescent dye. b) Incubation of the stationary phase obtained in stage a) under conditions which are suitable for the extension of 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 not suitable for removing NTs * which are not incorporated in a complementary strand. d) Detection of the individual NT * molecules built into complementary strands by measuring the signal which is characteristic of the respective fluorescent dye, at the same time determining the relative position of the individual fluorescent signals on the reaction surface. e) cleavage of the groups leading to the termination with the fluorescent dyes from the NTs * attached to the complementary strand to produce unlabeled (NTs or) NSKFs f) washing the stationary phase obtained in step e) under conditions which are suitable for removing the groups leading to termination with the fluorescent dyes Repetition of steps a) to f), if necessary several times, the relative position of individual NSKFs on the reaction surface and the sequence of these NSKFs being determined by specific assignment of the fluorescence signals detected in step d) in successive cycles at the respective positions to the NTs. 2. Surface for the immobilization of single-stranded NSKFs according to claim 1 characterized in that the NSKFs can be immobilized in a random arrangement on the surface, the density of the immobilized NSKF molecules being between 10 and 1,000,000 per 100 µm ² . 3. A flat surface according to claim 1, which consists of glass, plastic, gel or ceramic. 4. The method according to claim 1, in which in the detection step (d) the following types of detection are used: wide-field epifluorescence microscopy, laser scanning Fluorescence microscopy, TIRF microscopy. 5. The method of claim 1, wherein the concentration of individual sequence-specific Primer during hybridization (annealing) is between 10 pmol / l and 100 µmol / l. 6. A method according to claim 1 characterized in that it is for SNP analysis is used and a sequence-specific primer to identify each SNP site in the entire sequence is used. 7. A method according to claim 6 characterized in that the number of parallel to analyzing SNP sites is more than 2 and a sequence-specific for each SNP site Primer is used. 8. A method according to claim 1 characterized in that the overall sequence follows NSK species includes: genomic DNA and its descendants, mRNA populations or derived cDNA populations. 9. Primer according to the above claims, wherein primer one or more Primer populations represent a primer population to a section in the The entire sequence is complementarily sequence-specific and the primer molecules in one Primer population have identical sequence composition. 10. Primer according to claim 9, the length of which is between 10 and 100 NT.