DE102006007778B3 - Method for analyzing multiplex mixtures consisting of nucleic acid amplification products with overlapping detection regions - Google Patents

Abstract

The present invention relates to the field of molecular biological nucleic acid analysis. In particular, the invention relates to a method for genotyping nucleic acids, in particular for genotyping single base exchanges ("Single Nucleotide Polymorphisms", SNPs), microsatellites ("Short Tandem Repeats", STRs) or deletion and insertion polymorphisms (DIPs) in nucleic acids from amplicon mixtures which result from multiplex nucleic acid amplification procedures. The invention also relates to a kit with which the method according to the invention can be carried out.

Description

The The present invention relates to the field of molecular biology Nucleic acid analysis. In particular, the invention relates to a method for genotyping of nucleic acids, in particular for the genotyping of single base exchanges ("Single Nucleotide Polymorphisms ", SNPs), microsatellites ("Short Tandem Repeats ", STRs) or deletion and insertion polymorphisms (DIPs) in nucleic acids Amplification mixtures consisting of multiplex nucleic acid amplification result. Furthermore, the invention relates to a kit with which the inventive method carry out leaves.

In molecular biology nucleic acid analysis will be different detected in the sequence of DNA. Within homologous DNS sections between different species or individuals of a species (Population) these turn out to be longer defined sequence differences, single base exchanges ("Single Nucleotide Polymorphisms ", SNPs), sequence inversions, chromosome translocations or DNA deletions or -Insertionen. The latter go with a difference in length The investigated DNA region and can be subdivided again into simple DNA deletions and insertions ("deletion insertion polymorphisms", DIPs) or repetitive ones DNA elements such as minisatellites, microsatellites, SINE, LINE, telomeres. Diagnostically particularly important representatives of repetitive DNA elements are microsatellites ("Short Tandem Repeats ", STRs). STRs are variable sections of DNA that result from direct, tandem-like repetitions from 1-10 Base pairs (bp) exist (Butler, 2005). The number of repeat units of STRs can vary greatly between individuals of a species, so that an STR gene locus within a population often with more is represented as 4 alleles. In contrast, SNPs and DIPs usually only represented by two alleles (biallelic). In exceptions up to 4 alleles occur in SNPs. By simultaneous analysis multiple genetically uncoupled STRs (approximately 8-15) or SNPs and DIPs (ca. 30-60) can z. B. unique molecular genetic identification patterns (MI, "Genetics Fingerprint") to distinguish between individuals. These have become particularly enforced in forensics (perpetrator and genetic databases, forensic trace analysis), for parentage analysis (paternity report), for chimerism analysis after bone marrow transplantation, in tumor diagnostics ("Loess of heterozygosity ", LOH) and in animal and plant breeding (eg geographical origin and genealogical descent, inbreeding control, distinction from Plant varieties). For reasons of privacy will be for human MI exclusively DNA variations without phenotypic shaping used. The predominant However, a majority of molecular diagnostic applications involve DNA variations, the causative or indirectly to a phenotype can be obtained. An example is the detection of pathogens or transgenic organisms, tumor diagnosis, diagnosis of Hereditary diseases or diseases with genetic disposition that Pharmacogenetics or the characterization of performance features referenced in animal and plant breeding.

An important process step for the detection of genetic variations consists in the targeted duplication (amplification) of the selected DNA sequences in order to reach the sensitivity threshold of a downstream physico-chemical analysis system (principle of selective signal amplification). The most widely used technology for DNA amplification is the polymerase chain reaction (PCR) ( US 4,683,195 ). In this enzyme chemical method, the predetermined Zielseguenzen which z. , Species-specific sequences or genetic variations, from which DNA sample material is selectively amplified using thermostable DNA-dependent DNA polymerases. The substrate specificity of these enzymes presupposes that there is a single-stranded DNA template and a double-stranded DNA region located thereon, which is formed by base-pairing between the DNA template and a so-called PCR primer. The PCR primer is a synthetic oligonucleotide (at least 6, usually 15-40 bases) and must have a free 3'-hydroxyl group, in which, in the presence of deoxyribonucleoside triphosphates and magnesium ions, the synthesis of the new DNA strand into 5 '→ 3'-direction takes place. By combining two PCR primers into a PCR primer pair whose individual primers can each bind to a template strand of the DNA target sequence and with respect to their orientations in the 3 'direction, the exponential amplification of the DNA is carried out in a so-called locus-specific PCR intervening DNS region. The PCR is a cyclic process, each cycle consists of at least two temperature steps, the melting of the double-stranded DNA at about 90-95 ° C and the annealing and extending the primer at about 50-75 ° C. For this purpose, thermal cyclers are used. If the amplificate of a locus-specific PCR contains an SNP, its genotyping must include a further allele-specific step, such as, for example, "Allele Specific Single Base Extension" (SBE), DNA sequence analysis (eg, by Sanger statistical chain termination or by Pyrosequencing ^™ ), "allele-specific DNA hybridization,""allele-specific DNA probe technology" (eg. Fluorescence resonance energy transfer probes for real-time PCR) or "Oligospecific Ligation Assay" (OLA). Alternatively, an allele-specific PCR can be carried out, in which two primers each have an allele-specific sequence at their 3 'ends DNA variation (DIP or SNP) and in combination with one or two further locus-specific primers lead to allele-specific amplicons (eg "Amplification Refraction Mutation System", ARMST ^™ , EP 0 332 435 B2 or tetraprimer ARMS, Ye et al., 2001). The amplification of ribonucleic acids (RNA), e.g. B. for the detection of RNA-containing viruses or messenger RNA (mRNA) and their variations (eg, SNPs or splice variants), succeeds by the RNA before the PCR by reverse transcription by means of a reverse transcriptase in one to the RNA Sequence complementary DNA (cDNA) is rewritten.

Next The PCR are the person skilled in other methods for locus or allele-specific Amplification of nucleic acid regions known, which can also be carried out isothermally in part (see Reviews by Schweitzer and Kingsmore, 2001; Demidov and Broude, 2004). To call are u. a. "Nucleic Acid Sequence Based Amplification "(NASBA; Simpkins et al., 2000)," Ligase Chain Reaction "(LCR; Barany, 1991; in variations as a detection method also Padlock probes called), "loop-mediated Isothermal Amplification of DNA "(LAMP, Notomi et al., 2000) and "Multiplex Ligation-dependent probe amplification "(MLPA; Schouten et al., 2002; Langerak et al., 2005), "Beach Displacement Amplification "(SDA; Little et al., 1999), "Exonential Amplification Reaction "(EXPAR; van Ness et al., 2003) and "Rolling Cycle Amplification "(RCA; Lizardi et al., 1998).

to Genotyping of the DNA amplificates produced in this way subsequently become suitable detection methods needed. In the case of species-specific DNA fragments or STRs and DIPs whose Allele by length differences represent the amplificates, this can be done in DNA electrophoreses. The most important and currently most sensitive electrophoretic methods for the STR and DIP analysis is based on denaturing DNA capillary gel electrophoresis in combination with fluorescence detection of the single-stranded DNA amplicons. Therefor is ever a primer for the PCR during its chemical synthesis at its 5 'end with a fluorescent dye covalently linked and thus marked. Capillary gel electrophoreses are also possible miniaturize as "lab-on-a-chip" (Lin et al., 2005). Alternatively you can but also unlabelled STR and DIP amplificates in suitable flat gels by post-electrophoretic staining techniques based on dyes that selectively bind to DNA (eg. Ethidium bromide, Sybr Green, Sybr Gold), or by staining with Stains All or silver deposition (silver staining) are shown. For the genotyping of DIPs and SNPs are also different Methods described (see review article z. Syvänen, 2001 and Kwock, 2003). Here you have to so-called label-free methods (eg mass spectrometry or Surface plasmon resonance) of such be distinguished, the markers, z. In the form of fluorophores (eg fluorescence or fluorescence resonance energy transfer (FRET)), biotin (eg indirect colorimetric method via streptavidin and alkaline Phosphatase), haptens (eg in conjunction with an enzyme immunoassay) or Gold nanoparticles (absorption spectroscopy in combination with reductive Silver deposition on DNA hybridization chips; Clondiag, Jena, Germany) require. The above marks are made by organic chemical Synthesis covalently bound to PCR primers and DNA probes or z. During the SBE by means of fluorescence-labeled dideoxyribonucleotide triphosphates built-in.

critical is that the detection unit of the analyzer used a clear separation of the alleles by assignment of defined detection areas allowed. So dispose z. As fluorometers in sequencers or real-time PCR thermocylers currently over two to five color channels to appropriate Distinguish fluorophores. Alternatively, an allele specific Separation step done. Therefor are mass spectrometric and electrophoretic methods as well addressable microspheres ("Microspheres", "Beads") in combination with flow cytometers or DNA chips (DNS arrays) described. As addressing function for microspheres and DNS chips can for example, so-called "universal Sequence Tags "(Hirschhorn et al., 2000), d. H. specific or combinatorially generated and from each other different artificial sequences of about 6 to 50 Bases covalently attached to the 5'-end of SBE primers or allele-specific PCR primers. Other addressing functions in covalent bonding with primers are ligands such as biotin, digoxigenin (Roche Diognostics, Basel, CH) or other haptens in combination with streptavidin or specific antibodies or Derivatives of these. The skilled person is also known that surfaces of ferromagnetic particles with corresponding molecular interaction partners can be functionalized. Further modular combinations and variants for the separation of alleles or their detection is described by Syvänen (2001) and Kwock (2003).

In so-called multiplex amplifications, several target sequences are simultaneously amplified in biochemical one-pot methods, with the aim of enabling a simultaneous differential diagnosis of several species-specific DNA sequences or genetic polymorphisms such as SNPs, DIPs or STRs. In principle, such a procedure saves time and money. Of particular importance, however, are multiplexing applications for STRs, DIPs, and SNPs at ge medical questions in which a small initial amount of DNA, their degree of degradation or the presence of PCR inhibitors are often limiting for the success of genotyping. Further specimens with limited DNA output represent archaeological and wild biological samples as well as amniotic fluid biopsies or maternal blood samples for prenatal diagnosis.

As explained above is the simultaneous analysis especially in the generation of MIs a minimum number of genetically uncoupled STRs, DIPs or SNPs required. Variable STRs based on repeating units from 3 to 7 base pairs represent the current forensic standard for DNS databases and trace analysis (Butler, 2005). In multiplex PCR will be this often combined with DIPs in Amelogeningen, which the Sex determination (Nakahori et al., 1991). In the separation the PCR product mixtures (amplificates) of DIPs and in particular Of highly variable STRs in capillary gel electrophoresis must always a clear assignment of the individual amplificates to the corresponding ones Guaranteed originals (original loci). For this purpose may in the multiplex PCR the different DIP and STR loci with the same fluorescent label no overlaps between each other in the expected product sizes, d. H. the resulting amplification products with the same fluorescent label have to different lengths which make it possible to to separate the generated amplified products electrophoretically and via their variations in length attributable to the source alleles. They are therefore regarding theirs Amplificate size, d. H. their amplification product length, arranged one after the other. This means that in multiplex genotyping of STRs using the same fluorescent label the smallest potentially expected allele of the second STR locus must always be larger as the biggest allele of the first, the smallest allele of the third STR locus greater than the largest of the second. this leads to inevitably that the PCR products of the second or third STR locus of a Color range alleles with a length from 200-400 Have bases and more. The implementation of such an analysis but only possible although the starting DNA is present in at least this length. Otherwise receives you do not have a PCR product for these alleles. This often applies to forensic traces with degraded DNS too. The suitability of capillary gel electrophoresis for multiplex STR analysis in the trace range is thus limited by the space requirement of the individual STR-Loki and the number of usable color channels (currently 3-4, one color channel is always for an internal length standard reserved), which are available to distinguish between fluorescent colors. In contrast to the above, only in the case in which the amplificates of an STR locus with a different fluorescent label are provided as the amplificates of a second (or further) Locus, a unique mapping of overlapping (equal length) amplicons possible, as long as the number of different fluorescent labels used is not the number of color channels of the detector. The illustrated conditions define the detection areas for the single STR-Loki in this analysis method.

The major drawbacks of all genotyping methods described so far are that they consume DNA sample material that is no longer available for later STR analysis, and that they can analyze only a limited number of different loci per reaction batch simultaneously. Thus, z. For example, the most common alleles of the current 8 STR-Loki of the German DNA Database of the Federal Criminal Police Office and 13 of the Combined DNA Index System (CODIS) of the Federal Bureau of Investigations (FBI, USA) are not in a trackable multiplex PCR below 300 Bases are shown. Additional limitations arise from the fact that the excitation of the fluorophores often takes place with lasers of defined excitation wavelength, resulting in different sensitivities for the individual color markers. For trace analysis and similar applications of STR analysis, therefore, only particularly sensitive color markings are suitable, which further limits the multiplexing capability of the prior art process. Similar problems regarding the genotyping of multiple variable gene loci in multiplexing methods are also found in other analysis methods for SNPs, such as e.g. As mass spectrometry or Pyrosequencing ^TM given.

In In view of the above-described prior art, it is Object of the invention, simple, fast and inexpensive process which overlap the genotyping of themselves in a detection area Loki in multiplex amplification mixtures allow.

The The object is defined by what is stated in the present patent claims objects solved.

The explain the following figures The invention.

1 : Schematic representation of a multiplex PCR with overlapping detection areas for sexing and for generating a molecular identification pattern in forensics. Schematically represented are the detection areas of the individual Genloki in base pairs (bp, vertical bars and with numbers), which by the ge given primer pairs, the color coding and the smallest or largest alleles described in the literature are defined. In the lower part of the figure, the Genloki are the respective color labels (6FAM: 6-carboxyfluorescein, HEX: 4,7,2 ', 4', 5 ', 7'-hexachloro-6-carboxyfluorescein) and restriction endonucleases (REN), whose interfaces in the 5 'ends of the color-labeled primers were incorporated by chemical synthesis.

2 : Genotyping of a multiplex PCR with overlapping detection areas for sexing and for generating a molecular identification pattern in forensics. Schematically shown are electropherograms of the size range 65-150 base pairs of in 1 shown multiplex PCR, which were generated by means of the sequencer ABI Prism 310 Genetic Analyzer (Applied Biosystems, Darmstadt, Germany). The amplification products were electrophoretically separated without restriction digestion (A), after digestion with EcoRI (B) and after digestion with EcoRV (C). The Genloki (AC) are shown with their detection areas (open bars in B and C) above the electropherograms. The apparent fragment lengths differ from the theoretical by about 2 bp (see AM2 in C and 1 ). Abbreviations used are 6FAM (6-carboxyfluorescein), HEX (4,7,2 ', 4', S ', 7'-hexachloro-6-carboxyfluorescein) and RFE (Relative Fluorescence Units).

The As used herein, "PCR" or "polymerase chain reaction" refers to a process for primer-based DNA amplification.

The here used terms "RT-PCR" or "reverse Transcriptase PCR "or RT-polymerase chain reaction refer to a method for primer-based RNA amplification.

For this a particular messenger RNA (mRNA) is first transformed by means of a reverse Transcriptase in complementary Rewritten DNS (cDNS). This is followed by a PCR.

Of the used herein "Nucleic Acid Sequence Based Amplification "(NASBA) refers to a method of nucleic acid amplification at constant temperature. The target sequence is RNA. The standard reaction approach contains T7 RNA polymerase, RNase H, reverse transcriptase (RT), DNA polymerase, ribonucleotide triphosphates, Deoxyribonucleotide triphosphates, a primer other than the complementary to the target RNA Nucleotides at its 5 'end carries the sequence of the T7 RNA polymerase promoter and one to be read RNA sequence complementary Counterprimer (Schweitzer and Kingsmore, 2001).

Of the terms used herein "LCR", "ligase Chain Reaction, OLA or oligonucleotide ligation Assay "designate a method of nucleic acid amplification, in which two oligonucleotides (1 and 2) are selected so that they have no gap bind directly next to each other on the same DNA strand. The in the reaction mixture linked ligase linked the two strands with formation of a phosphodiester bond. To an amplification it comes, if in the same reaction mixture two further oligonucleotides 3 and 4, which belong to the two oligonucleotides 1 and 2 complementary are (Schweitzer and Kingsmore, 2001).

Of the used herein "Exponential Amplification Reaction "(EXPAR) refers to an isothermal method of nucleic acid amplification. This is in a cycle of single-strand break through a "Nicking Endonuclease "and Polymerization by a DNA polymerase amplified the target sequence (van Ness et al., 2003).

Of the term used here "loop-mediated Isothermal Amplification of DNA "(LAMP) denotes a method of nucleic acid amplification at the same remaining temperature. The reaction mixture contains i.a. a DNA polymerase, Deoxyribonucleotide triphosphates and a set of four primers, the are constructed so as to differentiate six different ones Sites of the target sequence are complementary (Notomi et al., 2000).

Of the term used herein "alleles Specific Single Base Extension "(SBE) refers to a procedure for the extension of the program of the 3'-hydroxyl end a primer around a nucleotide base using a DNA polymerase.

The terms "STR", "Short Tandem Repeat", "Microsatellites", or "Simple Sequence" as used herein are synonymous and refer to variable portions of deoxyribonucleic acids (DNA) consisting of direct, tandem repeats of 1-10 base pairs (bp) (http://www.ncbi.nlm.nih.gov/projects/SNP/). STRs are also considered to be special forms of the deletion and insertion polymorphisms ("DIPs" or "InDels"), since, like them, they are accompanied by differences in length of the analyzed DNA segment. The standard for forensic applications today is STR-Loki with repeating units consisting of 3 bp (eg GAT, AAT), 4 bp (eg GATA, AAAC), 5 bp (eg TAATA, AAAGG), 6 bp (eg AGAGAT, AATACC) or 7 bp (eg AAAAACC, GGATTAA). In population studies, however, interruptions of the defined repeat units by insertions and deletions as well as SNPs are observed. Thus, the detection range for a particular STR locus includes all measurable fragment length differences ≥ 1 bp, which between the largest and the smallest observed alleles including this are possible.

The Here used terms "DIP" or "InDel" denote simple Deletion and insertion polymorphisms resulting in differences in length of about 1 bp to 300 bp, 1 bp to 200 bp, 1 bp to 100 bp, preferably from about 1 bp to 50 bp of the analyzed DNA section (http://www.ncbi.nlm.nih.gov/projects/SNP/).

The here used terms "SNP" or "single Nucleotide polymorphism "refers to DNA variations based on single base exchanges in the DNA sequence (http://www.ncbi.nlm.nih.gov/projects/SNP/).

Of the As used herein, "detection range" refers to the analytic usable range of a physicochemical process in which the products of DNA amplification or genotyping clearly can be detected. This area is overlapping, if z. B. after a multiplex amplification, the products of various amplified Genloki are not clearly distinguishable from each other or not clearly assigned, z. B. if the products a same fragment length exhibit.

Of the As used herein, "primer" refers to single-stranded DNA oligonucleotides, those with a single-stranded DNA or RNA template a specific hybridization under training a DNA double helix or DNA RNA double helix and enter free 3'-OH end have. This molecular primer-template structure is the starting molecule of a DNS- or RNA-dependent DNA polymerase suitable. Own the primers used in the PCR a length from 6-50 Bases, where sequence-specific PCR usually primers from about 15 bases require. Preferred primer lengths are 10-50, 12-50, 15-50, 15-40, 20-40, 15-35, 15-30 bases. For PCR amplification of the desired DNA target sequence two primers, i. H. a so-called primer pair, designed so that one primer of this primer pair is the DNA target sequence to be amplified in 5'-direction in one Flanked from 1 to 550, preferably 1 to 350 base pairs and there hybridized to the coding DNA strand, and the second Primer of the primer pair the DNA target sequence to be amplified in 3'-direction in one Flanked from 1 to 550, preferably 1 to 350 base pairs and there hybridized to the noncoding DNA strand.

Of the used herein "multiplex" in combination with PCR, LCR, RCA, NASBA, SDA, EXPAR and LAMP refer to methods of nucleic acid amplification, where using more than one, e.g. B. two, three or four different primer pair (s) in one reaction more as a, z. B. two, three or four, target sequence (s) to be amplified be amplified.

The term "fluorophore" as used herein refers to a chemical compound capable of emitting energy by fluorescence in an excited state. Fluorophores used in molecular genetic diagnosis include, for example: 6-carboxyfluorescein (6FAM), 6-carboxy-4'-5'-dichloro -2'-, 7'-dimethoxy-fluorescein (JOE), 4,7,2 ', 4', 5 ', 7'-hexachloro-6-carboxy-fluorescein (HEX), 5- and 6-carboxy-X -rhodamine (ROX), 6-carboxytetramethyl-rhodamine (TAMRA), 6-carboxy-4,7,2 ', 7'-tetrachloro-fluorescein (TET) or 2'-chloro-5'-fluoro-7', 8'-benzo-1,4-dichloro-6-carboxyfluorescein (NED) ( US 6,316,610 ).

The here used terms "LOH" or "Loss of Heterozygosity " the differential diagnosis of certain tumors whose cells differ from healthy cells through the loss of certain DIP, SNP or STR alleles differ.

Of the As used herein, "FRET" or "Fluorescence Energy Resonance Transfer" or "Förster Energy Resonance Transfer" refers to radiation-free transfer of photon energy from an excited fluorophore (the donor) to another fluorophore (the acceptor) when the distance between both not more than 1-10 nm is.

The As used herein, "covalently linked", "covalently coupled "and" coupled "refers to covalent organic-chemical bonds.

The term "derivatives", "variations" or "polymorphisms" as used herein refers to nucleic acid sequences having one or more deletions, substitutions, additions, insertions and / or inversions. A deletion is a terminal or intramolecular loss of one or more nucleotides from a nucleotide sequence. Preferred are deletions of 1 to 10,000 nucleotides, 1 to 5,000 nucleotides, 1 to 1,000 nucleotides, 1 to 300 nucleotides and 1 to 50 nucleotides. A substitution is the replacement of one nucleotide for another, for example the replacement of an A for a T, C or G, a T for an A, C or G, a C for an A, T or G, a G against an A, T or C in a nucleotide sequence, wherein A is adenosine, T is thymidine, C is cytosine and G is guanosine. An addition is a terminal (at the 5 'and / or the 3' end of the nucleic acid) and an insertion by an intramolecular gain of one or more nucleotides in a nucleotide sequence. Preference is given to additions and insertions of 1 to 10,000 nucleotides, 1 to 5,000 nucleotides, 1 to 1,000 nucleotides, 1 to 300 nucleotides and 1 to 50 nucleotides. An inversion is the sequence reversal of a fragment of a nucleic acid within the nucleic acid, wherein the length of the inverted fragment is 1 to 10,000 nucleotides, 1 to 5,000 nucleotides, 1 to 1,000 nucleotides, 1 to 300 nucleotides and 1 to 50 nucleotides.

The inventive method based on multiplex nucleic acid amplification and detection techniques that involve more than one target sequence or nucleic acid variation in the form of species-specific DNS, SNPs, DIPs or STRs simultaneously be genotyped. There are two or more variable ones overlapping in a detection area Loki for species-specific DNS, such as STRs, DIPs, and SNPs, in a single reaction first allelic or locus specific amplified. In a following enzymatic, chemical or physical reaction step then the number of lengthwise overlapping Amplification products of genotyping Loki such kind selectively reduces that remaining amplification products subsequently with suitable detection method clearly allelic or locus specific can be analyzed.

• a) Mischen einer isolierten Probe, deren Nukleinsäuren genotypisiert werden soll, mit zwei oder mehr Primerpaaren, wobei jeweils ein Primer eines der zwei oder mehr Primerpaare oder die jeweils zwischen den Primern der zwei oder mehr Primerpaare liegende Nukleinsäuresequenz mindestens eine Nukleinsäuresequenzmodifikation umfasst,
• b) Amplifizieren der zu genotypisierenden Nukleinsäuren unter gleichzeitiger Erzeugung von zwei oder mehr verschiedenen Amplifikaten im Reaktionsgemisch mittels der zwei oder mehr Primerpaare, wobei die Amplifikate die mindestens eine Nukleinsäuremodifikation umfassen,
• c) Verteilen des Reaktionsgemischs aus Schritt b) auf zwei oder mehrere Reaktionsgefäße,
• d) Inkubieren der in den zwei oder mehr Reaktionsgefäßen vorliegenden Reaktionsgemische mit verschiedenen Nukleinsäure-spaltenden Enzymen, Enzymgemischen und/oder Chemikalien und/oder physikalische Behandlung der in den zwei oder mehr Reaktionsgefäßen vorliegenden Reaktionsgemische, wobei die Nukleinsäure-spaltenden Enzyme, Enzymgemische und/oder Chemikalien und/oder die physikalische Behandlung in Abhängigkeit von den Nukleinsäuresequenzmodifikationen der Amplifikate ausgewählt werden, so dass in den zwei oder mehr Reaktionsgefäßen vorliegenden Reaktionsgemischen jeweils nur ein definiertes Amplifikat aus einem Gemisch von Amplifikaten mit überlappenden Längenbereichen durch die enzymatische, chemische und/oder physikalische Behandlung nicht gespalten wird und die anderen Amplifikate durch die enzymatische, chemische und/oder physikalische Behandlung an der Nukleinsäuremodifikation gespalten werden, und
• e) Nachweisen der aus Schritt d) erhaltenen nicht gespaltenen Amplifikate.

The present invention relates in a first aspect to a method for genotyping nucleic acids, the method comprising the following steps:

• a) mixing an isolated sample whose nucleic acids are to be genotyped with two or more primer pairs, wherein in each case one primer of one of the two or more primer pairs or the nucleic acid sequence respectively lying between the primers of the two or more primer pairs comprises at least one nucleic acid sequence modification,
• b) amplifying the nucleic acids to be genotyped while simultaneously producing two or more different amplicons in the reaction mixture by means of the two or more primer pairs, the amplicons comprising at least one nucleic acid modification,
• c) distributing the reaction mixture from step b) to two or more reaction vessels,
• d) incubating the reaction mixtures present in the two or more reaction vessels with various nucleic acid-cleaving enzymes, enzyme mixtures and / or chemicals and / or physical treatment of the reaction mixtures present in the two or more reaction vessels, wherein the nucleic acid-cleaving enzymes, enzyme mixtures and / or Chemicals and / or the physical treatment depending on the nucleic acid sequence modifications of the amplificates are selected so that in the two or more reaction vessels present reaction mixtures only one defined amplificate of a mixture of amplificates with overlapping length ranges by the enzymatic, chemical and / or physical treatment is not cleaved and the other amplificates are cleaved by enzymatic, chemical and / or physical treatment at the nucleic acid modification, and
• e) detecting the non-cleaved amplicon obtained from step d).

By the evaluation of the detection result of the uncleaved amplicons, takes place afterwards the genotypic assignment of the amplificates to their original loci. The genotypic assignment of the amplificates to their original loci may also be done by prior to the detection in step e) of the method according to the invention a separation of the from step d) of the method according to the invention obtained uncleaved amplicons occurs.

at the isolated sample may be biological or non-biological Act on the sample. If it is a non-biological sample, includes nucleic acid-containing Material previously in contact with the non-biological sample has come. For example, it is the biological Sample around tissue samples like skin, or body fluids like blood, saliva, Serum, semen, vaginal fluids and the same. The samples can microbial, plant, animal or human origin. The sample may also contain deoxyribonucleic acid (DNA) previously obtained from biological material or ribonucleic acid (RNA).

at the nucleic acids to be genotyped may be deoxyribonucleic acid (DNS) or ribonucleic acid (RNA), with DNA being preferred. If the nucleic acid is RNA, takes place before the nucleic acid amplification a reverse transcription of the RNA in cDNA. If the nucleic acid is RNA the reverse transcription and nucleic acid amplification is preferably carried out by means of an RT-PCR.

at the inventively used For example, it can be used to amplify nucleic acids a PCR, RT-PCR, LCR, RT-LCR, NASBA, SDA, EXPAR, ARMS, or Tetraprimer ARMS as defined above act. Further A method of amplifying nucleic acids for use in the method of the invention useful are known to the skilled person. The implementation of these methods is known to the skilled person and depends on the corresponding standard protocols.

In an embodiment The present invention provides an isolated sample having two or more different primer pairs for a locus-specific multiplex PCR for genotyping two or more multiple STRs, and / or DIPs and appropriate reagents and a method of amplifying nucleic acids. In the case of STRs is a locus-specific PCR the preferred amplification method. After execution of the method for nucleic acid amplification the generated amplicons are present in the reaction mixture.

The Assignment of the amplificates to the individual according to the method of the invention Genloki to be genotyped is then preferably carried out by the Separation and detection of the amplificates in a denaturing Kapillargelelektrophorese, wherein the detection of the amplified by a detectable mark with which they are preferably provided are done. The labeling of the amplificates takes place according to the invention by one primer of each primer pair at its 5 'end or within the primer sequence, provided that the activities of the enzymes involved DNA polymerases and restriction endonucleases does not inhibit, with a Fluorescent dye is covalently coupled and thus marked. The fluorescent dye can be, for example, 6-FAM, TET, HEX, JOE or Be NED. By this primer also contains the corresponding amplified Product the dye marking and can thus by means of suitable Detector units are detected. In addition to the mentioned markings the primer or amplificate are further nucleic acid labeling options for the person skilled in the art such as biotin, haptens, addressing sequences and the like as well other physical detection methods for nucleic acid amplicons such as fluorescence, Fluorescence polarization, chemiluminescence, absorption spectroscopy, colorimetry, Mass spectroscopy, known as Surface Plasmon Resonance.

by virtue of the selection of primer sequences, the selection of color markers and the expected Amplifikatgrößen contains the reaction mixture of Multiplex PCR at least two to "n" different locus-specific products, which overlap in their detection areas, where "n" is the one in the reaction amplified to genopicting Loki corresponds to products with overlapping Lead detection area (that is, "n" is maximum exactly the number of Genloki amplified in the reaction). Wear Amplificates that are the same length and / or same or overlapping Claim detection areas, the same dye marking, can these amplicons are not separated in fluorescence detector analysis be detected. According to the inventive method therefore for distinguishing the latter overlapping amplification products, that come from the two or more genotyping loci, respectively the sequences of the locus-specific primers which are detectable at their 5 'ends Label, e.g. the dye marking, carry, with a specific Nucleic acid sequence modification provided. Also included by this nucleic acid sequence modification present in the primer the corresponding amplified product, the nucleic acid sequence modification.

In a preferred embodiment the nucleic acid sequence modification occurs by introducing of base exchanges or extensions their 5 'ends with the recognition sequences (cleavage sites) for various restriction endonucleases (Williams, 2003). Particularly suitable here are restriction endonucleases of type II, since their recognition and cleavage site are identical. The restriction endonucleases can for example EcoRV (recognition / cleavage sequence: 5'-GATATC-3 ') or EcoRI (recognition / cleavage sequence: 5'-GAATTC-3 '). Other restriction endonucleases and corresponding specific nucleic acid sequence modifications to introduction Restriction endonuclease cleavage sites, according to the present Invention useful are known to the skilled person. If the cleavage sites in the 5'-end of the marked It is also important that the restriction endonucleases are integrated their activity too at the end of a double-stranded DNS can develop.

In a further preferred embodiment may be the specific nucleic acid sequence modification be a recognition site for "nicking" endonucleases. When using "nicking" endonucleases, which catalyze a single-strand break (Williams, 2003) is the recognition sequence to orient so that the DNS strand is split, to which the detectable label, e.g. the dye label, coupled is.

Possibly can naturally in the sequence of fluorescence-labeled primers or within suitable regions of the amplificates occurring restriction sites for restriction or "Nicking" endonucleases at Design of the primer pairs for the multiplex PCR according to the invention be used. In general, however, it is crucial that on the one hand each individual locus-specific PCR product with overlapping Detection area assigned a clearly defined interface becomes (i.e., two to "n" different), which on the other hand the detection behavior of the other products within the multiplex-nucleic acid amplification mixture is not detrimental affected. For example, the one locus-specific amplificate assigned restriction site also not, z. Naturally, in overlapping Amplificate of another locus occur.

in the Connection to the multiplex nucleic acid amplification becomes the amplification mixture according to the number on products with overlapping Detection areas divided into 2 to "n" different reaction vessels. If the specific nucleic acid tag amplificates in introduction of restriction endonuclease cleavage sites, each reaction vessel is filled with a specific restriction endonuclease or a mixture of "n-1" specific restriction endonucleases incubated so that in each vessel only one specific fluorescently labeled amplification product from Product mixture with overlapping Detection area remains while the color markings of the other products cleaved endonucleolytically become. When using mixtures of restriction endonucleases may also be a sequential incubation with the restriction endonucleases occur when the restriction endonucleases different reaction buffer conditions need or have different temperature optima. However, they are also endonucleases which cut PCR directly in the buffer (eg EcoRI, EcoRV, KpnI). Following the endonucleolytic cleavages the unchanged at their 5 'ends covalently color-labeled reaction products of the individual reaction mixtures in denaturing capillary gel electrophoreses separated and by means of the capillary gel electrophoresis unit existing detection units, which detect the color marking can, clearly genotyped.

The Selection of the process step for the selective reduction of the number the overlapping in the detection area Amplificates with the same detectable label (eg fluorescence) The loci to be genotyped are made dependent on the ones in the modified ones Primers of the two or more primer pairs introduced specific nucleic acid sequence modifications, z. B. the imported Restriction endonuclease. In this case, a Mixture of "n-1" specific restriction endonucleases used. For example, in the multiplex amplification, if three genotyping loci (eg, STR A, STR B, STR C) are amplified be overlapping Detection areas can, in the 5 'region of the detectably marked, z. B. fluorescently labeled, primer of the first primer pair (for amplification from STR A) as modification z. B. an EcoRV restriction site, as described above become. In the 5'-range the detectably marked, z. B. fluorescently labeled primers of the second primer pair (for the amplification of STR B) can be used as Miodifikation z. B. an EcoRI restriction site are inserted. In the 5'-range of the detectable marked, z. B. fluorescently labeled primer of the third primer pair (for the amplification of STR C) can be used as a modification z. B. a HindIII Restriction interface inserted become. It should be noted that the inserted restriction sites in the 3 'direction of the fluorescence label are located. After the amplification reaction the amplicon mixture is distributed to three reaction vessels. In the first reaction vessel takes place subsequently a restriction digestion with the restriction enzymes EcoRV and EcoRI. In this case, the cleavage of the fluorescent dye units takes place the amplificates of STR A and STR B. Only the amplicons of STR C remain intact and can be detected fluorimetrically. In the second reaction vessel takes place according to a restriction digestion with the restriction enzymes EcoRV and HindIII. In this case, the cleavage of the fluorescent dye units takes place the amplicons of STR A and STR C. Only the amplicons of STR B remain intact and can be detected fluorimetrically. In the third reaction vessel takes place a restriction digestion with the restriction enzymes EcoRI and Hind III. In this case, the cleavage of the fluorescent dye units takes place the amplicons of STR B and STR C. Only the amplicons of STR A remain intact and can be fluorimetric be detected.

In a further embodiment of the present invention are used for the selective treatment of Multiplex PCR amplificates with overlapping Detection areas, d. H. for the specific cleavage of the dye label the overlapping and thus interfering with detection step Amplificates, Instead of Restriction Endonucleases for Specific DNA Modification (s) specific endonucleases, DNA N-glycosylases and / or AP-DNA lyases which are often described as DNA repair enzymes. For that will be According to the invention, the primers, which are provided with the detectable mark, with specific Nucleic acid sequence modifications provide the recognition sequences and / or cleavage sites for (a) certain DNA modification (s) specific endonucleases, DNA N-glycosylases and / or AP DNA lyases.

Specific nucleic acid sequence modifications useful in accordance with the method of the invention and allowing enzymatic cleavage of the detectable label from the amplicons are, for example, the DNA base modifications urea, 5,6-dihydroxythymine, thymine glycol, 5-hydroxy-5-methylhydantone, uracil glycol, 6 Hydroxy-5,6-dihydrothimine and methyltartronylurea. These DNA base modifications are selectively cleaved by endonuclease III from Escherichia coli. Endonuclease III has both DNA-N-glycosylase and AP-DNA lyase activity with respect to these substrates (Chaudhry and Weinfeld, 1995). Another specific nucleic acid sequence modification consists in the introduction of a Inositolrestes in the primer sequence, by means of the "nicking" endonuclease V from the organism Thermotoga maritima specifically two bases in 3 'direction of Inositolrestes leads to enzymatic cleavage of the DNA (Huang et al., 2002).

Further Alternatives to restriction endonucleases are well known to those skilled in the art (See, for example, Williams (2003)). Examples of Specific Nucleic Acid Sequence Modifications the according to the method according to the invention useful are recognition and / or cleavage sites for recombinant endonucleases, that by site-directed mutagenesis or random mutagenesis in their activity can be modified and adapted or so-called "Artificial Endonucleases " from genetic engineering mergers between a protein domain that has a specific DNA recognition mediates and a protein domain which has endonucleolytic activity. For the specific DNS sequence detection can e.g. B. sequence specific DNA binding domains for restriction endonucleases, bacterial regulatory sequences (eg: Cro, CAP, Helix-Turn-Helix-Motif) or unusual DNA conformations how triple helices are used (eg, complexes of RecA and Oligonucleotides). For DNA cleavage are z. B. protein domains Restriction endonucleases, topoisomerases and recombinases described and known to those skilled in the art.

In a further embodiment can be specific Nucleic acid sequence modifications the according to the method according to the invention are useful an enzymatic or chemical cleavage of the detectable label allow of the amplificates. For example, uracyl radicals introduced into the primer sequence become. DNA uracyl N-glycosylases cleave specifically in double-stranded DNA Uracyl residues to form apyrimidinic (AP) sites (Vaughan and McCarthy, 1998). The phosphodiester bonds of the AP sites can be cleaved chemically selective by means of alkali treatment (McHugh and Knowland, 1995; Sambrook et al., 1989), so that a Cleavage of the dye from the amplificate takes place. On the other hand allowed the derivatization of the primer sequence with AP sites also an enzymatic Cleavage of the dye from the amplificate by means of suitable AP-DNA lyases.

the Persons skilled in the art are aware of further specific nucleic acid sequence modifications. the according to the inventive method are useful a chemical cleavage of the detectable label from the amplicons cause. For example, you can 5'-amino-3'-deoxynucleoside Phorsporamidite to Synthesis of oligonucleotides with acid-labile 5'-P-N-3 'bonds can be used. (Shchepinov et al., 2001). Another method for site-specific endonucleolytic Cleavage of DNA is catalyzed by metal ions DNA cleavage (Williams, 2003). This is mediated by oligonucleotides, the target sequence specific double or triple helices form, or DNA binding proteins or corresponding protein domains the Sequence specificity. According to the present Invention can Thus, oligonucleotides can be used with the primer, the bears the detectable mark, train specific double or triple helices. Next, the specific nucleic acid sequence modifications Be DNA sequences for DNA binding proteins or corresponding protein domains confer sequence or binding specificity. The DNA cleavage and thus the cleavage of the dye from the amplificate takes place here chemically by metal ion complexes, for example ethylenediaminetetraacetic (EDTA) iron or o-phenanthroline copper covalently attached to the oligonucleotides or DNA binding proteins are coupled.

In a further embodiment can be specific Nucleic acid sequence modifications the primer takes place, which is a physical cleavage of the dye from the amplificate. Can do this PCR primers using photochemically unstable base analogues or oligonucleotide synthesis building blocks, for example, biotin (Li et al., 1999) or o-nitrobenzyl-CE-phosphoramidite (Wenzel et al., 2003), d. H. be provided that under the influence of UV light adjacent Phosphordiesterbindungen to split selectively. Further photochemically unstable conjugates between Oligonucleotides and peptides are described by Olejnik et al. (1999) wherein the peptide portion, for example, as hapten for detection by means of antibodies can serve or carry a dye marking.

to Genotyping according to the invention Amplification products from multiplex PCR mixtures with overlapping detection regions can be used above specific nucleic acid sequence modifications (derivatizations) of PCR primers also in such a way combined, that is a combination of postamplificatory enzymatic, chemical and / or photochemical (physical) Procedure to be performed got to.

Syvänen (2001) explicitly describes the multi-level and modular nature of various methods for genotyping SNPs, with alternative embodiments for certain analysis steps. The multiplexing capability of said methods is often limited by the number of non-overlapping detection areas, ie only multiplex amplificates whose lengths do not overlap can be uniquely detected. Covalent modifications of locus- or allele-specific primers are used both for detection (eg fluorescent dyes) and for locus- or allele-specific separation steps (eg biotin or addressing sequences).

Further embodiments of the present invention to increase the multiplexing ability by incorporation of alternative amplificate labels which are selective according to the invention should be removed, as well as with regard to alternative amplification and detection methods. It is crucial that during the Analysis of a DNA intermediate, ie a locus-specific amplicon, present a covalent attached label for fractionation and / or detection and as a selective substrate of an enzymatic, chemical and / or can serve physical cleavage. The specific nucleic acid sequence modifications (i.e., recognition or cleavage sequences for enzymes or base analogs, respectively) for the enzymatic, chemical and / or physical cleavage become selective while introduced the primer synthesis and serve as a label for the enzymatic, chemical and / or physical process step for selective reduction the number of overlapping lengths Amplificates of the loci to be genotyped. For the restriction endonucleases described above and repair endonucleases must thereby double-stranded DNA (dsDNA) or for certain Recombinases DNA Trippelhelices present.

To It should be noted that the inventive method in its analytical approach in principle different from other methods for the detection of SNPs and DIPs, which also includes the DNA amplification method with a subsequent Treatment of amplification products with appropriate enzymes is used (eg "Restriction Fragment Length Polymorphism ", RFLP; Reilly and Thomas, 1980; Ye et al., 2003; Till et al., 2004). About the Analysis of e.g. by restriction endonucleases (RFLP) or others Enzyme-digested reaction products in this process is the Presence or absence of naturally amplified in the Sequence occurring recognition sequence (s) for the enzyme used and characterized on the genotype of the sequence, the z. B. contains an SNP, closed. Ie. the DNA variation to be detected is associated with this case with a changed one Recognition sequence for a restriction endonuclease.

SAFFROY et al. (2002) further describe a method for the establishment of RFLPs if a corresponding allele-specific recognition sequence is natural only partially available. In this case, a primer in the 5 'direction in the immediate vicinity of analyzing DNA sequence polymorphism with its sequence at the 3 'end by base exchange (s) of the natural Sequence changed so is that (1) an enzymatic primer extension is possible and (2) simultaneously one in 3 'direction partial restriction endonuclease restriction site in the area of the DNA sequence polymorphism to be analyzed. Similarly US 2005/0214840 A1 describes a method for multiplex genotyping of SNPs using restriction endonucleases whose cleavage site in the 5'-range of one Amplification primer was integrated. This primer is located also in the 5'-direction moved nearby of the SNP and the amplification downstream cleavage, in particular using enzymes of the type IIS, serves an allele-specific detection reaction for the SNP. All of these methods are in the cited literature, if she for Multiplex applications are used, as one-pot reactions executed, d. H. no two or more reaction mixtures are formed post-amplification. About that In addition, these methods are not used to separate amplification mixtures with overlapping Detection areas used.

In contrast, are According to the invention, the specific modified DNA amplification products, their modification treated accordingly enzymatically, chemically and / or phasically were, for the selected one Detection method for genotyping just not available anymore, so not detectable because it eliminates from the multiplex approach according to the invention so that only the remaining amplification products, by the enzymatic, chemical and / or physical treatment not be influenced, analyzed and thus genotyped.

In a further embodiment, the inventive method can also be applied to single-stranded DNA products z. B. a multiplex SBE for the detection of SNPs apply. In this case, for example, "n" are amplified in a locus-specific nucleic acid amplification reaction using different loci containing, for example, SNPs 'Ends are labeled with biotin extended at their 3' ends with allelepezifischen, differently fluorescently labeled dideoxyribonucleotide. In a further step, the reaction mixture is purified from "n" different, allelepezifisch extended single-stranded SBE primers with streptavidin and distributed to "n" different reaction vessels. For each reaction vessel, two primers are now pipetted, which are complementary to the two allele-specifically extended SBE primers only a specific SNP locus. After DNA hybridization, only the double-stranded hybridization products are selectively protected from digestion with a single strand-specific DNA endonuclease (Till et al., 2004). All other allele-specifically extended SBE primers of the multiplex approach are degraded and thus the double labeling of the SBE primer with fluorophores and biotin is abolished. The genotyping of the individual SNPs is carried out after a second purification step using streptavidin via two-color fluorescence detection of the individual reaction mixtures.

In a modification of this embodiment can genotyping all SNPs of the multiplex approach using a single Color marking done. Therefor For example, the reaction mixture of this multiplexed SBE will double for the detection of "n" SNPs Number "2n" of reaction vessels distributed. All further steps take place analogously with the difference that in each case only one primer, which is complementary to an allele-specific extended SBE primer one specific SNP locus is used. Thus, per approach only an allele-specific extension product before digestion a single-stranded DNA endonuclease protected. This arrangement allows the genotyping of SNPs from complex Multiplex amplifications using optically simpler and thus especially cheaper Fluorometer. The selective cleavage of single-stranded SBE products can also done by so-called chemical endonucleases. It is about in this case to specific oligonucleotides, with a specific Target sequence can form a DNA double helix by DNA-DNA hybridization. The Cleavage occurs chemically through metal ion complexes, mostly ethylenediaminetetraacetic acid (EDTA) iron or o-phenanthroline copper covalently attached to the oligonucleotides coupled (Williams, 2003).

One Another aspect of the present invention is a kit for carrying out the inventive method, wherein the kit comprises two or more primer pairs for carrying out the Multiplex amplification according to the method of the invention includes. The primers include the corresponding primer pairs each DNS sequences that allow the desired target sequence (Loki) to amplify by the primer flanking the target sequence Hybridize DNA at a distance of about 50 to 350 base pairs. One of the two primers of the primer pair is according to step a) of the method according to the invention labeled, for example, with a fluorescent dye as above described. This labeled primer additionally contains a specific nucleic acid sequence modification, as described above, wherein for each locus a different specific nucleic acid sequence modification must exist and thus with the help of two or more different ones enzymatic, chemical and / or physical treatment steps in two or more separate reactions each one of the loci on the enzymatic, chemical and / or physical treatment from the multiplex mixture is eliminated and a remaining unchanged amplification product in the subsequent analysis method as described above characterized, d. H. genotyped, will. Furthermore, the kit according to the invention may comprise reaction vessels, in which the reaction mixture from step c) of the method according to the invention can be distributed. Furthermore, the kit according to the invention can be used accordingly of the to be carried out Multiplex method suitable for nucleic acid amplification Reagents such as buffers, nucleotides and enzymes, e.g. B. DNA polymerases, and suitable reference nucleic acid (s) in suitable containers.

• – Identifizierung oder Ausschluss von Tätern in der Gerichtsmedizin und forensischen Spurenanalytik.
• – Nachweis der genealogischen Abstammung bei Mensch und Tier (Vaterschaften, Stammbaumanalysen).
• – Geografischer Herkunftsnachweis von Tieren und Pflanzen sowie die Genotypisierung zur Unterscheidung von Pflanzensorten.
• – Überwachung der Biodiversität oder Inzucht in Zuchttierbeständen.
• – Archäologie und Evolutionsstudien bei Menschen und anderen Spezies. Hierfür werden u.a. archäologische und fossile Artefakte biologischen Ursprungs genotypisiert.
• – Chimärismusanalyse nach Knochenmarkstransplantationen.
• – Pränatale Diagnostik unter Verwendung fötaler DNS aus dem Blutplasma oder dem Fruchtwasser der werdenden Mutter.
• – Differenzialdiagnostik von Hefe- und Bakterienstämmen, welche sich in variablen STRs unterscheiden. Die Anwendungsgebiete umfassen die Fermentationsindustrie, die Lebensmittelanalytik sowie den Nachweis von human-, tier- und pflanzenpathogenen Stämmen.
• – Genotypisierung biallelischer SNPs und DIPs durch elektrophoretische Verfahren.
• – Differenzialdiagnostik bestimmter Tumoren, welche sich von gesunden Zellen im Verlust bestimmter DIP- oder STR-Allele unterscheiden („Loss of Heterocygosity", LOH).

The method according to the invention for the analysis of complex multiplex mixtures for nucleic acid amplification products with overlapping detection regions for genotyping can be used for all known fields of application of STR, SNP and DIP analysis. Of particular importance are multiplex applications when the starting amount of nucleic acid is limiting. In particular, genotyping to distinguish individuals of a species in the following areas should be mentioned:

• - Identification or exclusion of forensics practitioners and forensic trace analysis.
• - Proof of genealogical descent in humans and animals (paternity, pedigree analyzes).
• - Geographical evidence of origin of animals and plants and genotyping to distinguish plant varieties.
• - Monitoring of biodiversity or inbreeding in breeding stock.
• - Archeology and evolutionary studies in humans and other species. Archaeological and fossil artefacts of biological origin are genotyped for this purpose.
• - Chimerism analysis after bone marrow transplantations.
• - Prenatal diagnosis using fetal DNA from the blood plasma or the amniotic fluid of the expectant mother.
• - Differential diagnosis of yeast and bacterial strains, which differ in variable STRs. The areas of application include the fermentation industry, food analysis and the detection of human, animal and phytopathogenic strains.
• - Genotyping of biallelic SNPs and DIPs by electrophoretic methods.
• - Differential diagnosis of certain tumors, which differ from healthy cells in the loss of certain DIP or STR alleles ("Loss of Heterozygosity", LOH).

The present invention will be described below using the example of a combined STR and DIP analysis using locus-specific multiplex PCR and genotyping by denaturing capillary gel electrophoresis and fluorescence detection. The analytical resolution of the complex multiplex PCR mixture succeeds by postamplificatory fractionation, whereby an amplification product to be analyzed is freed from all other amplification products which interfere with the detection, which overlap in the detection area, because the fluorescence labels necessary for the detection detection are replaced by an amplification product Cleaved restriction endonuclease.

EXAMPLE

Multiplex PCR and genotyping of five polymorphs STR loci and a DIP locus with overlapping detection areas. For sex determination in humans is a DIP of 6 bp in Range of the amelogenin gene suitable, which in each case a copy on both sex chromosomes in meiotic non-recombining areas is localized (AM2, Nakahori et al., 1991). It concerns with this DIP around the sequence 5'-AAAGTG-3 ', which are the base pairs 332 to 337 of the Y-specific gene copy with accession number GenBank: M55419 and between bases 331 and 332 of the X-specific gene copy with accession number GenBank: M55418 is missing. To create a genetic fingerprints in humans were used as examples autosomal STR markers of the CODIS gene database standard THO1 (accession numbers: UniSTS: 156170; GenBank: NT_009237), TPOX (accession numbers: UniSTS: 240638; GenBank: NT_033000), CSF1PO (accession numbers: UniSTS: 156169; GenBank: NT_029289), D3S1358 (accession numbers: UniSTS: 148226; GenBank NT_022517) and DSS818 (Accession numbers: UniSTS: 54700; GenBank: NT_034772).

Using the cited sequence information, the references Butler et al. (2003) and Krenke et al. (2002) and the software VectorNTI (Invitrogen, Karlsruhe, Germany), the following PCR primers were designed: AMFT3-H (SEQ ID NO: 1) (5'-HEX-TTT GAATTC CCTGGGCTCTGTAAAGAA-3 '; artificial sequence appendix in bold text, Underlined for EcoRI), AMR3 (SEQ ID NO: 2) (5'-ATCAGAGCTTAAACTGGGAAGCTG-3 '), D3RT1-F (SEQ ID NO: 3) (5'-6FAM-TT GATATC ATGAAATCAACAGAGGCTTGC-3'; bold type, site underlined for EcoRV), D3F1 (SEQ ID NO: 4) (5'-ACTGCAGTCCAATCTGGGT-3 '), D5RT3-F (SEQ ID NO: 5) (5'-6FAM-TTT GAATTC AGCCACAGTTTACAACATTTGTATCT-3'; in bold type, sequence for EcoRI underlined), D5FT3 (SEQ ID NO: 6) (5'-GGTGATTTTCCTCTTTGGTATCC-3 '), THOFT3-F (SEQ ID NO: 7) (5'-6FAM-TTT GAATTC CTGTTCCTCCCTTATTTCCC- 3 '; boldface artificial suffix, underlining EcoRI site), THOR3 (SEQ ID NO: 8) (5'-GGGAACACAGACTCCATGGTG-3'), TPFT3-H (SEQ ID NO: 9) (5'-HEX-TTT GAATTC TTAGGGAACCCTCACTGAA TG-3 '; in bold type, sequence for EcoRI underlined), TPR3 (SEQ ID NO: 10) (5'-GTCCTTGTCAGCGTTTATTTGC-3 '), CSFT1-H (SEQ ID NO: 11) (5'-HEX-TT GATATC ACAGTAACTGCCTTCATAGATAG- 3 '; boldface artificial sequence appendix, site underlined for EcoRV) and CSR1 (SEQ ID NO: 12) (5'-GTGTCAGACCCTGTTCTAAGTA-3'). The synthesis of the oligonucleotides was carried out by an external service laboratory. Standard molecular genetic techniques were performed according to Sambrook et al. (1989). All chemicals and consumables were used in pa quality or PCR quality (free of DNA, proteases and nucleases). Genomic DNA (GDNS) from volunteer donors was obtained from cheek swabs (with sterile cotton swabs) and blood samples using the kit NucleoSpin ^® Tissue kit (Macherey & Nagel, Düren) and NucleoSpin ^® Blood (Macherey & Nagel, Düren) prepared according to manufacturer's instructions. Absorbance measurements at 260 nm were used as reference methods for DNA quantification by means of UV / VIS spectroscopy (BioPhotometer, Eppendorf Hamburg, Germany) and real-time PCR measurements with the "Quantifiler ^™ Human DNA Quantification Kit" (Applied Biosystems, Darmstadt, Germany The genomic DNA supplied with the kit was used as the reference DNA The 25 μL volume multiplex PCR approach consisted of 1.5 mM MgCl ₂ , 200 nM dNTPs, 1-fold concentrated JumpStart reaction buffer (Sigma & Aldrich, Freiburg), 1.25 Unit Jump Start ^® Taq DNA polymerase (Sigma & Aldrich, Freiburg), 5.0 ng of genomic DNA and the following oligonucleotides with their Endkonzemtrationen in parentheses: AMFT3-H (0.4 uM) , AMRT3 (0.4 μM), D3RT1-F (0.2 μM), D3F1 (0.2 μM), D5RT1-F (0.6 μM), D5FT1 (0.6 μM), THOFT3-F (0 , 6 μM), THOR3 (0.6 μM), TPFT3-H (0.2 μM), TPR3 (0.2 μM), CSFT1-H (0.2 μM) and CSR1 (0.2 μM) PCR program consisted of an initi Denaturing for 2 min at 94 ° C, followed by 30 cycles of 20 s at 94 ° C, 1 min at 55 ° C and 30 s at 72 ° C and another 60 min at 60 ° C. A TC-512 thermal cycler (Techne AG, Burkhardtsdorf) was used. After the PCR, 9.6 μL each of the batch were incubated in separate reaction vessels containing 5 units each of the restriction endonucleases EcoRI (New England Biolabs, Frankfurt aM) and EcoRV (New England Biolabs, Frankfurt aM, Germany) for 1.0 h incubated at 37 ° C. This was followed by heat inactivation for 20 min at 82 ° C. Subsequently, the genotyping of the Genloki performed. For this purpose, the sequencer ABI ^Prism® 310 Genetic Analyzer (Applied Biosystems, Darmstadt, Germany) was used. 1 μL each of the PCR reaction or the corresponding restriction mixtures was mixed with 12.5 μL injection buffer consisting of 12 μL HiDi formamide (Applied Biosystems, Darmstadt, Germany) and internal length standard HD-400ROX (Applied Biosystems, Darmstadt, Germany), Denatured at 95 ° C for 5 min and rapidly cooled to 10 ° C. The sample injection was carried out at 15,000 V for 5 sec. The capillary gel electrophoresis was carried out with the polymer mixture POP-4 (Applied Biosystmens, Darmstadt, Germany) at 60 ° C., 15,000 V and 20 min under denaturing conditions according to the manufacturer's instructions. Evaluated with the software GenScan Analysis (Applied Biosystems, Darmstadt, Germany) using a specially prepared color matrix for the dyes 6-FAM, HEX and ROX according to the manufacturer. The electropherograms for a woman's DNA are in 2 shown.

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It follows a sequence listing according to WIPO St. 25. This can from the official publication platform downloaded from the DPMA.

Claims (39)

Method for genotyping nucleic acids, wherein the method comprises the following steps: a) mixing one isolated sample, their nucleic acids genotyped, with two or more primer pairs, where one primer each of the two or more primer pairs or the each between the primers of the two or more primer pairs nucleic acid sequence at least one nucleic acid sequence modification includes, b) amplifying the genotyping nucleic acids under simultaneous generation of two or more different amplicons in the reaction mixture by means of the two or more primer pairs, wherein the Amplificates comprising at least one nucleic acid modification, c) Distributing the reaction mixture from step b) to two or more Reaction vessels, d) Incubate the present in the two or more reaction vessels Reaction mixtures with different nucleic acid-cleaving enzymes, enzyme mixtures and / or chemicals and / or physical treatment in the two or more reaction vessels present Reaction mixtures, wherein the nucleic acid-cleaving enzymes, enzyme mixtures and / or chemicals and / or the physical treatment depending from the nucleic acid sequence modifications the amplificates selected be present so that in the two or more reaction vessels Reaction mixtures in each case only one defined amplificate from one Mixture of amplificates with overlapping length ranges by the enzymatic, chemical and / or physical treatment is not cleaved and the other amplificates by the enzymatic, chemical and / or physical treatment at the nucleic acid modification be split, and e) detecting the products obtained from step d) uncleaved amplicons. The method of claim 1, wherein the sample is a biological Sample is, in particular a tissue sample such as skin, or a body fluid such as blood, saliva, serum, seminal fluid or vaginal fluid, or isolated DNA, RNA or cDNA. The method of claim 2, wherein the sample is microbial, of plant, animal or human origin. Method according to one of the preceding claims, wherein amplifying the nucleic acids by enzymatic methods such as polymerase chain reaction (PCR), Reverse transcription polymerase chain reaction (RT-PCR), ligase chain Reaction (LCR), Reverse Transcriptional Ligase Chain Reaction RT (LCR), Nucleic Acid Sequence Based Amplification (NASBA), Strand Displacement Amplification (SDA), Exponential Amplification Reaction (EXPAR), Amplification Refractory Mutation System (ARMS) or Tetraprimer Amplification Refractory Mutation System (Tetraprimer-ARMS). A method according to any one of the preceding claims, wherein a primer of one of the two or more primer pairs and the amplicons produced by means of the two or more primer pairs has a detection base a label, for example a fluorescent label, a colorimetrically detectable label and / or an immunologically detectable label comprises / include. The method of claim 5, wherein the fluorescent label 6-carboxyfluorescein (6-FAM), 4,7,2 ', 4', 5 ', 7'-hexachloro-6-carboxy-fluorescein (HEX), 6-carboxy-4,7,2 ', 7'-tetrachloro-fluorescein (TET), 6-carboxy-4'-, 5'-dichloro-2'-, 7'-dimethoxy-fluorescein (JOE), 6-carboxy-X-rhodamine (ROX), 6-carboxytetramethyl-rhodamine (TAMRA) or 2'-chloro-5'-fluoro-7 ', 8'-benzo-1,4-dichloro-6-carboxyfluorescein (NED), the colorimetrically detectable label is a biotin / strepavidin-alkaline Phosphatase conjugate and the immunologically detectable label is a hapten. Method according to one of claims 5 or 6, wherein the detectable Labeling covalently with the 5 'end the primer is connected. Method according to one of the preceding claims, wherein the nucleic acid sequence modification an enzymatic, chemical and / or physical cleavage of Amplificates and / or cleavage of the detectable label allowed. The method of claim 8, wherein the nucleic acid sequence modification is a recognition and cleavage site for endonucleases. The method of claim 9, wherein the endonuclease a restriction endonuclease, an endonuclease III, a nicking endonuclease, a DNA N-glycosylase and / or an apyridinic site (AP) DNA lyase is. The method of claim 8, wherein the nucleic acid sequence modification allows the enzymatic generation of apyrimidinic (AP) sites. The method of claim 11, wherein the nucleic acid sequence modification is a uracyl residue that is transformed by DNA into a uracyl-N-glycosylase apyrimidinic (AP) site is transferred. The method of claim 8, wherein the nucleic acid sequence modification a DNA split allowed under the influence of UV light. The method of claim 13, wherein the nucleic acid sequence modification a photochemically unstable base analogue such as biotin, an o-nitrophenyl and / or an o-nitrobenzyl derivative is. Method according to one of the preceding claims, wherein at least one nucleic acid sequence modification occurs in each of the generated amplicons is located in the sequence area of the template nucleic acid to be genotyped, to the primer comprising at least one nucleic acid sequence modification, hybridized, or wherein in the area to be amplified between the two primers of a primer pair a recognition and cleavage site for endonucleases so that enzymatic cleavage of the amplicons and / or the cleavage of the detectable label is made possible. Method according to one of the preceding claims, wherein the two or more different amplificates that overlap one another Have length range, have two or more different nucleic acid sequence modifications, a selective cleavage of the amplificates and / or cleavage of allow detectable labeling of amplified products. Method according to one of the preceding claims, wherein the nucleic acids to be genotyped at least one single base exchange, for example a single nucleotide polymorphism (SNP), a microsatellite, for example a short tandem repeat (STR) and / or a deletion and insertion polymorphism (DIP). Method according to one of the preceding claims, wherein the two or more generated amplicons have different nucleic acid sequence modifications which include the enzymatic, chemical and / or physical Cleavage of the amplificates and / or cleavage of the detectable label allow and enzymatic, chemical and / or physical Cleavage of the amplificates and / or cleavage of the detectable label in Step d) of claim 1 takes place simultaneously or sequentially. Method according to one of the preceding claims, wherein prior to detecting the uncleaved amplicon in step e) a separation of the amplificates by means of electrophoresis, in particular by means of denaturing Kapillargelelektrophorese done. Use of a primer comprising a detectable Mark and in 3'-orientation thereof at least one nucleic acid sequence modification to carry out a method according to a the claims 1-19. Use according to claim 20, wherein the detectable Label a fluorescent label, a colorimetrically detectable Tag or immunologically detectable tag. Use according to claim 21, wherein the fluorescent label is 6-carboxyfluorescein (6-FAM), 4,7,2 ', 4', 5 ', 7'-hexachloro-6-carboxy-fluorescein (HEX), 6-carboxy-4,7,2', 7'-tetrachloro-fluorescein (TET), 6-carboxy-4'-, 5'-dichloro-2'-, 7'-dimethoxy-fluorescein (JOE), 6-carboxy-X-rhodamine (ROX), 6-carboxytetramethyl-rhodamine (TAMRA) or 2'-chloro-5'-fluoro-7 ', 8'-benzo-1,4-dichloro-6-carboxyfluorescein (NED), the colorimetrically detectable label a biotin / streptavidin-alkaline phosphatase conjugate and the immunologically detectable label a hapten is. Use according to claim 20, wherein the nucleic acid sequence modification an enzymatic, chemical and / or physical cleavage of detectable mark allowed. Use according to claim 23, wherein the primer is a Detection and cleavage site for Includes endonucleases. Use according to claim 24, wherein the endonuclease a restriction endonuclease, an endonuclease III, a nicking endonuclease, a DNA N-glycosylase and / or an apyrimidinic site (AP) DNA lyase is. Use according to claim 23, wherein the nucleic acid sequence modification allows the enzymatic generation of apyrimidinic (AP) sites. Use according to claim 26, wherein the nucleic acid sequence modification is a uracyl residue that is transformed by DNA into a uracyl-N-glycosylase apyrimidinic (AP) site is transferred. Use according to claim 23, wherein the nucleic acid sequence modification a DNA split allowed under the influence of UV light. Use according to claim 28, wherein the nucleic acid sequence modification a photochemically unstable base analogue such as biotin, an o-nitrophenyl or an o-nitrobenzyl derivative is. Kit to carry out of the method according to any one of claims 1-19 comprising in a suitable container two or more primer pairs, each one primer of a primer pair a detectable label and in 3'-orientation thereof at least one Having nucleic acid sequence modification. The kit of claim 30, wherein the detectable label a fluorescent label, a colorimetrically detectable label or an immunologically detectable label. The kit of claim 31, wherein the fluorescent label 6-carboxyfluorescein (6-FAM), 4,7,2 ', 4', 5 ', 7'-hexachloro-6-carboxy-fluorescein (HEX), 6-carboxy-4,7,2 ', 7'-tetrachloro-fluorescein (TET), 6-carboxy-4'-, 5'-dichloro-2'-, 7'-dimethoxy-fluorescein (JOE), 6-carboxy-X-rhodamine (ROX), 6-carboxytetramethyl-rhodamine (TAMRA) or 2'-chloro-5'-fluoro-7 ', 8'-benzo-1,4-dichloro-6-carboxyfluorescein (NED), the colorimetrically detectable label is a biotin / strepavidin-alkaline Phosphatase conjugate and the immunologically detectable label a hapten is. The kit of claim 30, wherein the nucleic acid sequence modification an enzymatic, chemical and / or physical cleavage of detectable mark allowed. A kit according to claim 33, wherein the primer is a recognition and cleavage site for Includes endonucleases. The kit of claim 34, wherein the endonuclease is a Restriction endonuclease, an endonuclease III, a nicking endonuclease, a DNA N-glycosylase and / or an apyrimidinic site (AP) DNA lyase is. The kit of claim 33, wherein said nucleic acid sequence modification allows the enzymatic generation of apyrimidinic (AP) sites. The kit of claim 36, wherein the nucleic acid sequence modification is a uracyl residue that is transformed by DNA into a uracyl-N-glycosylase apyrimidinic (AP) site is transferred. The kit of claim 33, wherein said nucleic acid sequence modification allows DNA cleavage under the action of UV light. The kit of claim 38, wherein the nucleic acid sequence modification a photochemically unstable base analogue such as biotin, an o-nitrophenyl or an o-nitrobenzyl derivative.