WO2007095923A1 - Method for analysing multiplex-mixtures comprising nucleic acid amplification products having overlapping detection areas - Google Patents

Abstract

The invention relates to the field of molecular biological nucleic acid analysis. The invention also relates to a method for genotyping nucleic acids, in particular for genotyping SNPs (Single Nucleotide Polymorphisms'), microsatellites ('Short Tandem Repeats', STRs) or deletion and insertion polymorphisms (DIPs) in nucleic acids from amplification mixtures resulting in multiplex-nucleic acids amplification methods. The invention also relates to a kit enabling said invention to be carried out.

Description

 Method for analyzing multiplex mixtures consisting of nucleic acid amplification products with overlapping detection regions

The present invention relates to the field of molecular biology nucleic acid analysis. In particular, the invention relates to a method for genotyping nucleic acids, in particular for single nucleotide polymorphisms (SNPs), short tandem repeats (STRs) or deletion and insertion polymorphisms (DIPs) in nucleic acids from amplified mixtures, the resulting from multiplex nucleic acid amplification. Furthermore, the invention relates to a kit with which the inventive method can be carried out.

In molecular biology nucleic acid analysis differences in the sequence of DNA are detected. Within homologous DNA segments between different species or individuals of a species (population) these are longer defined sequence differences, single nucleotide polymorphisms (SNPs), sequence inversions, chromosome translocations or DNA deletions or insertions. The latter are associated with a difference in length of the investigated DNA region and can in turn be subdivided into simple DNA deletions and insertions ("deletion insertion polymorphisms", DIPs) or repetitive DNA elements such as minisatellites, microsatellites, SBSfE, LINE, telomeres. Diagnostically most important members of repetitive DNA elements are Short Tandem Repeats (STRs), which are variable sections of DNA consisting of direct, tandem repeats of 1-10 base pairs (bp) (Butler, 2005) the repeat units of STRs can vary widely between individuals of one species, so that an STR gene locus within a population is often represented by more than 4 alleles, in contrast SNPs and DIPs are usually represented by only two alleles (biallelic) In exceptional cases, up to 4 alleles can be found in SNPs, which can be identified by the simultaneous analysis of several non-linked STRs (about 8-15) or SNPs and DIPs (about 30-60), for example, with unique molecular genetic identification patterns (MI, " Genetic fingerprint ") to distinguish individuals. These have particularly prevailed in the Forensics (perpetrator and genetic databases, forensic trace analysis), for pedigree analysis, for chimerism analysis after bone marrow transplantation, in tumor diagnostics ("JLoss of Heterozygosity", LOH) and in animal and plant breeding (eg geographical origin and genealogical descent, For reasons of data protection, human MI uses only DNA variations without phenotypic characteristics, but the vast majority of molecular diagnostic applications concern DNA variations that can be causally or indirectly related to a phenotype Detection of pathogens or transgenic organisms, the tumor diagnosis, the diagnosis of hereditary diseases or diseases with genetic disposition, the pharmacogenetics and the characterization of performance characteristics in animal and plant breeding referenced.

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 commonly used technology for DNA amplification is the polymerase chain reaction (PCR) (US 4,683,195). In this enzyme chemical method, the predetermined target sequences, 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 in 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 ⁰ C exists. For this purpose, thermal cyclers are used. Does the amplicon contain a locus-specific PCR a SNP, it must for genotyping another allelepezifischer step such. 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 the real-time PCR) or "Oϊigospecific Ligation Assay" (OLA). Alternatively, an allele-specific PCR can be carried out in which two primers each contain an allele-specific sequence of the DNA variation (DIP or SNP) at their 3 'ends and in combination with one or two further locus-specific primers lead to allele-specific amplicons (eg. Amplification Refractory Mutation System ", ARMS ™, EP 0 332 435 B2 or Tetraprimer-AMS, Ye et al., 2001) Amplification of ribonucleic acids (RNA), eg for the detection of RNA-containing viruses or messengers RNA (mRNA) and its variations (eg SNPs or splice variants) can be achieved by transcription of the RNA by reverse transcription using reverse transcriptase into DNA complementary to the RNA sequence (cDNA).

Apart from the PCR, further methods for locus- or allele-specific amplification of nucleic acid regions are known to the person skilled in the art, some of which can also be carried out isothermally (see reviews by Schweitzer and Kingsmore, 2001, Demidov and Broude, 2004). To call are u. a. "Jellide Acid Sequence Based Amplification" (NASBA; Simpkins et al., 2000), "JLigase Chain Reaction" (LCR; Barany, 1991, also referred to as Padlock Probes in variations as a detection method), JLoop-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)," Strand Displacement Amplification "(SDA, LMe et al , 1999), Exponential Amplification Reaction (EXPAR), Rolling Cycle Amplification (RCA), Lizardi et al., 1998, and Felicase-Dependent Isothermal Amplification (Vincent et al. , 2004).

For the genotyping of the DNA amplificates thus generated, suitable detection methods are subsequently required. In the case of species-specific DNA fragments or STRs and DIPs whose alleles are represented by differences in length of the amplificates, this can be done in DNA electrophoreses. The most important and currently most sensitive electrophoretic method for STR and DIP analysis is based on denaturing DNA capillary gel electrophoresis combined with fluorescence detection of the single-stranded DNA amplicons. This will ever a PCR primer is covalently linked to a fluorescent dye at its 5 'end during its chemical synthesis and thus labeled. Capillary gel electrophoresis can also be miniaturized as an "off-on-a-chip" (Lin et al., 2005) Alternatively, however, unlabeled STR and DIP amplificates can be selectively prepared in suitable flat gels by post-electrophoretic staining techniques based on dyes bind to DNA (eg, ethidium bromide, Sybr Green, Sybr Gold), or by Stains All staining or silver staining (silver staining) For the genotyping of DIPs and SNPs also various methods are described (see review article eg. Syvänen, 2001 and Kwock, 2003), in which so-called label-free methods (eg mass spectrometry or surface plasmon resonance) have to be differentiated from those containing labels, eg in the form of fluorophores (eg fluorescence or Fluorescence Resonance Energy Transfer (FRET)), biotin (e.g., indirect colorimetric method via streptavidin and alkaline phosphatase), haptens (e.g., in conjunction with an enzyme immunoassay) or gold nanoparticles (absorption spectroscopy in combination with reductive silver deposition on DNA hybridization chips; Clondiag, Jena, Germany). The said markers are covalently bound by PCR to organic chemical synthesis to PCR primers and DNA probes or z. B. incorporated during the SBE by means of fluorescence-labeled dideoxyribonucleotide triphosphates.

The decisive factor is that the detection unit of the analyzer used permits a clear separation of the alleles by assigning defined detection ranges. To have z. For example, fluorometers in sequencers or real-time PCR thermocylers currently have two to five color channels to distinguish appropriate fluorophores. Alternatively, an allele-specific separation step can take place. For this purpose, mass spectrometric and electrophoretic methods as well as addressable microspheres ("microspheres", "beads") in combination with flow cytometers or DNA chips (DNA arrays) are described. As an addressing function for microspheres and DNA chips, for example, so-called "Universal Sequence Tags" (Hirschhorn et al., 2000), ie specific or combinatorially generated and different from each other artificial sequences of about 6 to 50 bases covalently bound to the 5 ' Other covalently linked covalent binding moieties to primers include ligands such as biotin, digoxigenin (Roche Diognostics, Basel, CH) or other haptens in combination with streptavidin or specific antibodies or derivatives of .RTM It is also known to the person skilled in the art that surfaces of ferromagnetic particles can be functionalized with corresponding molecular interaction partners modular combinations and variants for the separation of alleles or their detection are 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 multiplex applications for STRs, DIPs, and SNPs in forensic issues, where a low starting amount of DNA, its degree of degradation, or the presence of PCR inhibitors are often limiting to 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, the simultaneous analysis of a minimum number of genetically uncoupled STRs, DIPs or SNPs is required, in particular in the generation of MIs. Variable STRs based on repeating units of 3 to 7 base pairs represent the current forensic standard for DNA databases and trace analysis (Butler, 2005). In multiplex PCR, these are often combined with DIPs in the amelogenin gene, which are used for sexing (Nakahori et al., 1991). When separating the PCR product mixtures (amplificates) from DIPs and in particular from highly variable STRs in capillary gel electrophoresis, it is always necessary to ensure a clear assignment of the individual amplificates to the corresponding origin alleles (original loci). For this purpose, in multiplex PCR, the various DIP and STR loci with the same fluorescent label with each other must have no overlaps in the expected product sizes, ie the resulting amplification products with the same fluorescent label must have different lengths, which allow the generated amplificates electrophoretically separate and to be assigned to the source alleles via their length variations. They are therefore arranged one after the other with regard to their amplificate size, ie their amplification product length. This means that in multiplex genotyping of STRs using the same fluorescent label, the smallest potential allele of the second STR locus must always be larger than the largest allele of the first, the smallest allele of the third STR locus greater than the largest second. This inevitably leads to the PCR products of the second or third STR locus of a color range Alleles with a length of 200-400 bases and more have. The implementation of such an analysis is only possible if the starting DNA is present in at least this length. Otherwise, no PCR product is obtained for these alleles. This often applies to forensic evidence with degraded DNA. The suitability of Kapillargelelektrophorese for multiplex STR analysis in the trace range is thus limited by the space requirements of each STR Loki and the number of usable Farbkänale (currently 3-4, a color channel is always reserved for an internal length standard), the distinction of fluorescent colors are available. In contrast to the above, only in the case where the Amplifϊkate one STR locus are provided with a different fluorescent label than the amplificates of a second (or further) locus, a unique assignment of overlapping (equal length) amplificates is possible, as long as the Number of different fluorescent labels used does not exceed the number of color channels of the detector. The described framework conditions define the detection areas for the individual STR loci in this analysis method.

The major disadvantages 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 with respect to the genotyping of multiple variable genioki in multiplexing methods are also found in other analysis methods for SNPs, such as e.g. As mass spectrometry or pyrosequencing given.

In view of the state of the art outlined above, it is an object of the invention to provide simple, rapid and inexpensive methods involving the genotyping of allow overlapping loci in multiplex amplification mixtures in one detection area.

The object is achieved by the objects defined in the present patent claims.

The following figures illustrate the invention.

Fig. 1: Schematic representation of a multiplex PCR with overlapping detection areas for sex determination and for generating a molecular Idendifizmussters in forensics. Schematically represented are the detection regions of the individual genioki in base pairs (bp, vertical bars and with numbers), which are defined by the given primer pairs, the color coding and the smallest or largest alleles described in the literature. In the lower part of the figure, the Genioki are the respective color markers (6FAM: 6-carboxyfluorescein, HEX: 4, 7, 2 ', 4', 5 ', 7'-hexachloro-6-carboxy-fluorescein) and restriction endonucleases (REN), whose interfaces have been incorporated into the 5 'ends of the color-labeled primers by chemical synthesis.

Fig. 2: Genotyping of a multiplex PCR with overlapping detection areas for sex determination and for generating a molecular identification pattern in forensics. Schematically shown are electropherograms of the size range 65-150 base pairs of the multiplex PCR shown in Fig. 1, 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 EcόBl (B) and after digestion with EcoRV (C). The genioki (A-C) 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 or FIG. 1). Abbreviations used are 6FAM (6-carboxyfluorescein), HEX (4, 7, 2 ', 4', 5 ', T-hexachloro-6-carboxyfluorescein) and RFE (Relative Fluorescence Units).

The terms "PCR" or "polymerase chain reaction" as used herein refer to a method for primer-based DNA amplification.

The terms "RT-PCR" or "Reverse Transcriptase PCR" or "RT" Polymerase chain reaction refers to a method for primer-based RNA amplification. For this purpose, a specific messenger RNA (niRNA) is first transcribed by means of a reverse transcriptase into complementary DNA (cDNA). This is followed by a PCR.

The term "Nucleic Acid Sequence Based Amplification" (NASBA) as used herein refers to a method of nucleic acid amplification at steady temperature. "" The target sequence is RNA. "" The standard reaction contains T7 RNA polymerase, RNase H, reverse transcriptase (RT), DNA polymerase. Ribonucleotide triphosphates, deoxyribonucleotide triphosphates, a primer which carries, in addition to the nucleotides complementary to the target RNA, at its 5 'end the sequence of the T7 RNA polymerase promoter and a complementary primer complementary to the RNA sequence read (Schweitzer and Kingsmore, 2001).

The terms "LCR", "JLigase Chain Reaction", "OLA" or "Oligonucleotide Ligation Assay" as used herein refer to a method of nucleic acid amplification in which two oligonucleotides (1 and 2) are selected to be directly adjacent to one another without space Bind DNA strand. The ligase contained in the reaction mixture links the two strands to form a phosphodiester bond. An amplification occurs when two further oligonucleotides 3 and 4 are contained in the same reaction mixture, which are complementary to the two oligonucleotides 1 and 2 (Schweitzer and Kingsmore, 2001).

As used herein, JSxponential Amplification Reaction '' '' (EXPAR) refers to an isothermal method of nucleic acid amplification. In this case, the target sequence is amplified in a cycle of single-strand breakage by a "nicking endonuclease" and polymerization by a DNA polymerase (van Ness et al., 2003).

As used herein, the term "loop-mediated isothermal amplification of DNA" (LAMP) refers to a method of nucleic acid amplification at a constant temperature The reaction mixture contains, inter alia, a DNA polymerase, deoxyribonucleotide triphosphates and a set of four primers designed to are complementary to six different distinct sites of the target sequence (Notomi et al., 2000). The term "helicase-dependent isothermal amplification" as used herein refers to a method of nucleic acid amplification at a constant temperature The reaction mixture contains inter alia a DNA polymerase, deoxyribonucleotide triphosphates, two primers in an analogous arrangement as in the PCR, a helicase and a DNA single-strand binding protein ( Vincent et al., 2004).

The term "single base extension" (SBE) or "allele specific SBE" as used herein refers to a method for the template-specific extension of the 3'-hydroxyl end of a primer by one nucleotide base using a DNA polymerase.

The terms "STR", "Short Tandem Repeat \" Microsatellite ", 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 deletion and insertion polymorphisms ("DIPs" or "InDels"), since they are like these with length differences of the STR-Loki with repeat 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) However, in population studies interruptions of the defined repeat units by insertions and deletions as well as SNPs are observed Detection area for a specific STR locus all measurable fragment length differences> 1 bp, which are possible between the largest and the smallest observed allele including this.

As used herein, the terms "DIP" or "InDel" refer to simple deletion and insertion polymorphisms resulting in length differences 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 examined DNS section (http://www.ncbi.nlm.nih.gov/projects/SNP/).

The terms "SNP" or "single nucleotide polymorphism" as used herein refer to DNA variations based on single base exchanges in the DNA sequence (http://www.ncbi.nlm.nih.gov/projects/SNP/). As used herein, the term "detection range" refers to the analytically useful range of a physicochemical process in which the products of DNA amplification or genotyping can be uniquely detected, which range is overlapping if, for example, after multiplex amplification, the products of the different amplified genioki can not be clearly distinguished from one another or can not be unambiguously assigned, for example if the products have the same fragment length.

As used herein, the term "primer" refers to single-stranded DNA oligonucleotides that undergo specific hybridization with a single stranded DNA or RNA template to form a DNA double helix or DNA RNA double helix and have a free 3'-OH end. This molecular primer-template structure is suitable as a starting molecule of a DNA or RNA-dependent DNA polymerase.The primers used in the PCR have a length of 6-50 bases, with sequence-specific PCR usually require primers from about 15 bases 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, a so-called Primer pair, designed such that one primer of this primer pair flanks the DNA target sequence to be amplified in the 5 'direction at a distance of 1 to 550, preferably 1 to 350 base pairs and hybridizes there to the coding DNA strand, and the second Pr imer of the primer pair to be amplified DNA target sequence in the 3 'direction at a distance of 1 to 550, preferably 1 to 350 base pairs flanked and there hybridized to the non-coding DNA strand.

The term "multiplex" as used herein in combination with PCR, LCR, RCA, NASBA, SDA, EXPAR and LAMP refers to methods of nucleic acid amplification in which using more than one, eg two, three or four different primer pair (s) ) in a reaction batch more than one, eg, two, three or four, to be amplified target sequence (s) are 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-caiboxy-4,7,2 ', 7'-tetrachloro-flιιorescein (TET), 2'-Chloro-5'-fluoro-7', 8 ^l -benzo-l, 4-dichloro-6-carboxyfluorescein (NED) (US 6,316,610), N, N' - (dipropyl) -tetramethylindocarbocyanine (Cy3) or N, N - (dipropyl) tetramethylindodicarbocyanine (Cy5).

The terms "LOH" or "JLoss of Heterozygosity" used herein refer to the differential diagnosis of certain tumors whose cells differ from healthy cells by the loss of particular DIP, SNP or STR alleles.

The term "FRET" or "Fluorescence Energy Resonance Transfer" or "J Forster Energy Resonance Transfer" as used herein refers to the radiation-free transfer of photon energy from one excited fluorophore (the donor) to another fluorophore (the acceptor), when the distance between both is not more than 1-10 in.

The terms "covalently linked", "covalently coupled" and "coupled" as used herein refer 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 3' end of the nucleic acid) and an insertion by an intramolecular gain of one or more nucleotides in a nucleotide sequence. Preferred are 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 inversion 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 1000 nucleotides, 1 to 300 nucleotides and 1 to 50 nucleotides.

The method of the invention is based on multiplex nucleic acid amplification and detection techniques that simultaneously genotype more than one target sequence or nucleic acid variation in the form of species-specific DNA, SNPs, DIPs, or STRs. Two or more variable, overlapping in a Detelctionsbereich Loki for species-specific DNA, such as STRs, DIPs and SNPs, amplified in a single reaction initially allele or locus specific. In a subsequent enzymatic, chemical or physical reaction step, the number of lengthwise overlapping amplification products of the loci to be genotyped is then selectively reduced in such a way that the remaining amplification products can then be analyzed unambiguously allelically or locus-specifically using suitable detection methods.

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 lying in each case 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 with simultaneous generation of two or more different amplicons in the reaction mixture by means of the two or more primer pairs, the amplicons comprising the at least one nucleic acid modification, c) distributing the reaction mixture from step b) to two or more reaction vessels, d) incubating the in the two or more reaction vessels present reaction mixtures with different nucleic acid-cleaving enzymes, enzyme mixtures and / or chemicals and / or physical treatment of the present in the two or more reaction vessels reaction mixtures, the Nukl acid-cleaving enzymes, enzyme mixtures and / or chemicals and / or the physical treatment depending on the nucleic acid sequence modifications of the amplicons are selected so that present in the two or more reaction vessels Reaction mixtures only one defined amplificate from a mixture of Amplifϊkaten with overlapping length ranges by the enzymatic, chemical and / or physical treatment is not cleaved and the other amplificates are cleaved by the enzymatic, chemical and / or physical treatment at the nucleic acid modification, and e) Detecting the uncleaved amplicons obtained from step d).

By evaluating the detection result of the non-cleaved Amplifϊkate, then the genotypic assignment of the amplificates to their original loci. The genotypic assignment of the amplificates to their original loci can also be carried out by separating the non-cleaved amplificates obtained from step d) of the method according to the invention before the detection in step e) of the method according to the invention.

The isolated sample may be a biological or non-biological sample. If it is a non-biological sample, it includes nucleic acid-containing material that has previously come into contact with the non-biological sample. For example, the biological sample is tissue samples such as skin, or body fluids such as blood, saliva, serum, seminal fluid, vaginal fluids, and the like. The samples may be of microbial, plant, animal or human origin. The sample may also be previously deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) derived from biological material.

The nucleic acids to be genotyped may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), with DNA being preferred. If the nucleic acid is RNA, reverse transcription of the RNA into cDNA occurs prior to nucleic acid amplification. When the nucleic acid is RNA, reverse transcription and nucleic acid amplification are preferably carried out by RT-PCR.

The method according to the invention for the amplification of nucleic acids can be, for example, a PCR, RT-PCR, LCR, RT-LCR, NASBA, SDA, EXPAR, ARMS, helicase-dependent isothermal amplification or tetraprimer ARMS as defined above , act. Other methods of amplifying nucleic acids useful in the method of the invention are known to those skilled in the art. The implementation of this Method is known to the skilled person and depends on the corresponding standard protocols.

In one embodiment of the present invention, an isolated sample with two or more different primer pairs for a locus-specific multiplex PCR for the genotyping of two or more STRs, and / or DIPs and suitable reagents is added and carried out a process for the amplification of nucleic acids. In the case of STRs, locus-specific PCR is the preferred amplification method. After carrying out the process for Nuldeinsäureamplifϊkation the generated amplicons are present in the reaction mixture.

The assignment of the amplificates to the individual genotypes to be genotyped by the method according to the invention is then preferably carried out by the separation and detection of the amplificates in a denaturing capillary gel electrophoresis, the amplificates being detected by a detectable label with which they are preferably provided. According to the invention, the amplification is marked by one primer of each primer pair at its 5 'end or within the primer sequence, provided this does not inhibit the activities of the enzymes involved, DNA polymerases and restriction endonucleases. Covalently coupled with a fluorescent dye and labeled. The fluorescent dye may be, for example, 6-FAM, TET, HEX, JOE or NED. This primer also contains the appropriately amplified product, the dye label and can thus be detected by means of suitable detector units. In addition to the above-mentioned markings of the primers or amplificates, further nucleic acid labeling possibilities such as biotin, haptens, addressing sequences and the like as well as further physical detection methods for nucleic acid amplificates such as fluorescence, fluorescence polarization, chemiluminescence, absorption spectroscopy, colorimetry, mass spectroscopy, surface plasmon resonance are known to the person skilled in the art.

Because of the selection of primer sequences, the selection of color labels, and the expected amplicon sizes, the multiplex PCR reaction mixture will contain at least two to "n" different locus-specific products that overlap in their detection regions, with "n" of the gene to be genotyped in the reaction amplified Loki that results in products with overlapping detection range (ie, "n" corresponds at most exactly to the number of genioki amplified in the reaction.) Carry amplificates that are the same length and / or claim identical or overlapping detection regions, the same dye marking, these amplificates can not be detected separately in the fluorescence detector analysis. In accordance with the method according to the invention, therefore, to distinguish the latter overlapping amplification products which originate from the two or more loci to be genotyped, in each case the sequences of the locus-specific primers which carry the detectable label, eg the dye marking, at their 5 'ends specific nucleic acid sequence modification provided. By virtue of the nucleic acid sequence modification present in the primer, the correspondingly amplified product also contains the nucleic acid sequence modification.

In a preferred embodiment, the nucleic acid sequence modification is performed by introducing base exchanges or extensions of 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 sites are identical in each case. The restriction endonucleases may be, for example, üeoRV (recognition / cleavage sequence: 5'-GATATC-3 ') or EcoRI (recognition / cleavage sequence: 5'-GAATTC-3'). Additional restriction endonucleases and corresponding specific nucleic acid sequence modifications for introduction of restriction endonuclease cleavage sites useful in accordance with the present invention are known to those skilled in the art. Furthermore, if the cleavage sites are integrated at the 5 'end of the labeled primer, it is important that the restriction endonucleases can also exert their activity at the end of a double-stranded DNA.

In another preferred embodiment, the specific nucleic acid sequence modification may be a recognition site for jsficking endonucleases When using nicking endonucleases that catalyze a single strand break (Williams, 2003), the recognition sequence should be oriented so that the DNA strand is cleaved to which the detectable mark, eg the dye marking is coupled.

"Mcfo ^'" g - "- where appropriate primer labeled fluorescent or occurring within appropriate ranges of the amplified interfaces for restriction or may occur naturally in the sequence of. Endonucleases in the design of the primer pairs for the novel multiplex PCR are used generally here, however crucial, on the one hand, each one On the other hand, the locus-specific PCR product with overlapping detection region is assigned a clearly defined interface (ie two to "n" different) which, on the other hand, does not adversely affect the detection behavior of the other products within the multiplex nucleic acid amplification mixture also occur, for example, naturally, in the overlapping amplificate of another locus.

Following the multiplex nucleic acid amplification, the amplification mixture is divided into 2 to "n" different reaction vessels according to the number of products with overlapping detection regions If the specific nucleic acid tag of the amplicons consists in the introduction of restriction endonuclease cleavage sites, each reaction vessel is exposed to a specific restriction endonuclease or a mixture of "n-1" specific restriction endonucleases, so that only one specific fluorescence-labeled amplification product from the product mixture with overlapping detection region remains in each vessel, while the color markings of the other products are endonucleolytically cleaved off. When using mixtures of restriction endonucleases, a sequential incubation with the restriction endonucleases can also take place if the restriction endonucleases require different reaction buffer conditions or have different temperature optima. However, endonucleases are also known which cut PCR directly in the buffer (eg EcoKL, EcoKV, KpnT). Following the endonucleolytic cleavages, the covalently color-coded reaction products of the individual reaction batches, which are covalently color-coded at their 5 'ends, are separated in denaturing capillary gel electrophoreses and clearly genotyped by means of detection units present on the capillary gel electrophoresis unit which can detect the color coding.

The selection of the method step for the selective reduction of the number of amplificates overlapping in the detection region with the same detectable label (eg fluorescence) of the loci to be genotyped is done as a function of the specific nucleic acid sequence modifications introduced into the modified primers of the two or more primer pairs, e.g. The introduced restriction endonuclease cleavage sites. In this case, a mixture of "n-1" specific restriction endonucleases is used, for example, in the multiplex amplification, if three loci to be genotyped (e.g., STR A, STR B, STR C) having overlapping detection regions are amplified in the 5'-range of the detectable marked, z. B. fluorescence-labeled, primer of the first primer pair (for the amplification of STR A) as a modification z. For example, an EcoRV restriction site as described above may be included. In the 5 'region of the detectably marked, z. B. fluorescence-labeled, primer of the second primer pair (for the amplification of STR B) can be used as a modification z. B. an EcO BJ restriction site are inserted. In the 5 'region of the detectably marked, z. B. fluorescence-labeled, primer of the third primer pair (for the amplification of STR C) can be used as a modification z. B. a HmdlII restriction site are inserted. It should be noted that the inserted restriction sites are located in the 3 'direction of the fluorescent label. After the amplification reaction, the amplificate mixture is distributed to three reaction vessels. The first reaction vessel is then subjected to restriction digestion with the restriction enzymes EcόSCV and EcoRI. In this case, the cleavage of the fluorescent dye units of the amplificates of STR A and STR B takes place. Only the amplificates of STR C remain intact and can be detected fluorimetrically. In the second reaction vessel, a restriction digestion with the restriction enzymes EcoRV and HindIII takes place correspondingly. In this case, the cleavage of the fluorescent dye units of the amplificates of STR A and STR C takes place. Only the amplificates of STR B remain intact and can be detected fluorimetrically. In the third reaction vessel is a restriction digestion with the restriction enzymes EcoRI and HindIII. The cleavage of the fluorescent dye units of the amplificates of STR B and STR C is carried out. Only the amplificates of STR A remain intact and can be detected fluorimetrically.

In a further embodiment of the present invention, for the selective treatment of multiplex PCR amplificates with overlapping detection regions, i. H. for the specific cleavage of the dye label of the overlapping and thus in detection step interfering amplicons, instead of restriction endonucleases for (a) specific DNA Modifϊkation (s) specific endonucleases, DNA N-glycosylases and / or AP-DNA lyases used, often as DNA repair enzymes are described. For this purpose, according to the invention, the primers which are provided with the detectable label are provided with specific nucleic acid sequence modifications, the recognition sequences and / or cleavage sites for (a) specific DNA modification (s) are specific endonucleases, DNA N-glycosylases and / or AP-DNA. Lyases are.

Specific nucleic acid sequence modifications according to the method of the invention For example, the DNA base modifications include urea, 5,6-dihydroxythymine, thymine glycol, 5-hydroxy-5-methylhydantone, uracil glycol, 6-hydroxy-5,6-dihydrothimine, and allow enzymatic cleavage of the detectable label from the amplicons 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 an inositol residue into the primer sequence which specifically leads to enzymatic cleavage of the DNA by means of the ,, Mcfang "endonuclease V from the organism Uiermotoga maritima two bases in the 3 'direction of the inositol residue (Huang et al. , 2002).

Other 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 which are useful according to the method of the invention are recognition and / or cleavage sites for recombinant endonucleases which can be modified and adapted in their activity by site-directed mutagenesis or random mutagenesis or so-called "artificial endonucleases" consisting of genetic engineering fusions between a protein domain that mediates specific DNA recognition and a protein domain that has endonucleolytic activity For specific DNA sequence recognition, for example, sequence-specific DNA binding domains for restriction endonucleases, bacterial regulatory sequences (e.g., Cro, CAP , Helix-Turn-Helix-Motif) or unusual DNA conformations such as triple helices are used (eg complexes of RecA and oligonucleotides) For DNA cleavage, for example, protein domains of restriction endonucleases, topoisomerases and recombinases beschri just and known to the skilled person.

In another embodiment, specific nucleic acid sequence modifications useful in accordance with the method of the present invention may permit enzymatic or chemical cleavage of the detectable label from the amplicons. For example, uracyl residues can be introduced into the primer sequence. DNA uracyl N-glycosylases specifically cleave uracyl residues in double-stranded DNA to form apyrimidinic (AP) sites (Vaughan and McCarthy, 1998). The phosphodiester bonds of the AP sites can be cleaved thermally (WO99039001) or chemically selectively by means of alkali treatment (McHugh and Knowland, 1995, Sambrook et al., 1989), so that a cleavage of the dye from the amplificate. On the other hand, the derivatization of the primer sequence with AP sites also allows an enzymatic cleavage of the dye from the amplificate by means of suitable AP-DNA lyases.

The skilled worker is aware of further specific nucleic acid sequence modifications which are useful according to the inventive method to cause a chemical cleavage of the detectable label of the amplificates (see also EP 0828855B1). For example, 5'-amino-3'-deoxynucleoside phosphorporamidites can be used for the synthesis of oligonucleotides having acid-labile 5'-P-N-3 'bonds. (Shchepinov et al., 2001). Another method for site-specific endonucleolytic cleavage of DNA is by metal ion-catalyzed DNA cleavage (Williams, 2003). In this case, oligonucleotides which form specific double or triple helices with the target sequence or DNA binding proteins or corresponding protein domains mediate the sequence specificity. Thus, according to the present invention oligonucleotides can be used which form specific double or triple helices with the primer carrying the detectable label. Further, the specific nucleic acid sequence modifications may be DNA sequences that confer sequence specificity or binding specificity to DNA binding proteins or protein domains, respectively. The DNA cleavage and thus the cleavage of the dye from the amplificate takes place here chemically by metal ion complexes, for example ethylenediaminetetraacetic acid (EDTA) iron or o-phenanthroline copper, which are covalently coupled to the oligonucleotides or DNA binding proteins.

In another embodiment, specific nucleic acid sequence modifications of the primers may be made that allow for physical cleavage of the dye from the amplicon. For this purpose, PCR primers can be derivatized by means of photochemically unstable base analogues or oligonucleotide synthetic building blocks, for example biotin (Li et al., 1999) or o-nitrobenzyl-CE-phosphoramidite (Wenzel et al., 2003, DE 10108453B4), i. H. which selectively split adjacent phosphorodiester bonds under the action of UV light. Further photochemically unstable conjugates between oligonucleotides and peptides are described by Olejnik et al. (1999), whereby the peptide portion can serve for example as hapten for the detection by means of antibodies or can carry a coloring material marking.

For genotyping according to the invention of amplification products from multiplex PCR For mixtures with overlapping detection regions, the above-mentioned specific nucleic acid sequence modifications (derivatizations) of PCR primers can also be combined in such a way that a combination of the postamplificatory enzymatic, chemical and / or photochemical (physical) processes has to be carried out.

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 ranges, i. H. only multiplex amplificates whose lengths do not overlap can be clearly 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).

Other embodiments of the present invention for enhancing multiplexing capability are provided by incorporation of alternative amplificate labels to be selectively removed in accordance with the present invention, as well as alternative amplification and detection techniques. Crucial here is that during the analysis, a DNA intermediate, ie a locus-specific amplicon, is present, which contains a covalent attached label for fractionation and / or detection and can serve as a selective substrate of an enzymatic, chemical and / or physical cleavage. The specific nucleic acid sequence modifications (ie, enzymatic, chemical, and / or physical cleavage sequences) are selectively introduced during primer synthesis, thereby serving as a label for the enzymatic, chemical, and / or physical selective reduction step the number of lengthwise overlapping amplificates of the loci to be genotyped. For the above-described restriction endonucleases and repair endonucleases, double-stranded DNA (dsDNA) must be present or, for certain recombinases, DNA triple helices.

Other embodiments of the present invention for enhancing multiplexing capability result from the approach of selectively labeling a subset of unlabeled amplification products after multiplexing in separate reaction mixtures. Thus, the cleavage products of restriction endonucleases, preferably of type II, which single-stranded 5 'excess length, are enzymatically labeled by extending the DNA half-strand, which contains the 3'-hydroxyl end offset inside. This can be done after restriction digestion in a single base extension (SBEs) using a DNA-dependent DNA polymerase (EC2.7.7.7) and labeled nucleotide triphosphates or labeled chain-termination reagents, preferably 2 ', 3'-dideoxyribonucleoside triphosphates (ddNTPs), Che & Chen, 2004, US 2005/02148401, IAl) Other chain termination reagents are known to the person skilled in the art Examples of suitable labels are derivatives of dNTPs and ddNTPs with fluorophores (eg fluorescein-d (eg. d) NTP, TAMRA-d (d) NTP, Cy3-d (d) NTP, Cy5-d (d) NTP), biotin or radioactivity (alpha ³² P-dNTP or alpha ³² P-ddNTP, alpha ³³ P-dNTP or alpha ³³ P-ddNTP).

For multiplex PCR and subsequent exact genotyping of multiple STRs yielding amplification products with overlapping detection regions, one PCR primer per gene locus is extended at its 5 'end with the site for a particular restriction endonuclease, which naturally does not exist in the gene locus to be amplified is present or not associated with the polymorphism to be examined or interacts with its detection. If, for example, only one measuring device for the detection of fluorescein and only fluorescein-ddGTP for an SBE is available for the detection, it is possible with the incorporation of e.g. B. the restriction endonuclease sites of BamΗI (5'-G / GATCC-3 'interface, the slash symbolizes the cleavage site in the half-strand), BgIIl (5'-A / GATCT-3' interface, the slash symbolizes the cleavage site in the half-strand) , Bell (5'-T / GATCA-3 'intersection, the slash symbolizes the cleavage site in the half-strand), Eagl (5'-C / GGCCG-3' intersection, the slash symbolizes the cleavage site in the half-strand), PspOMl (5 'interface). -GZGGCCC-S ', the slash symbolizes the cleavage site in the half-strand) and Kasl (S'-G / GCGCC-S' interface, the slash symbolizes the cleavage site in the half-strand), BstEll (5'-G / GTNACC-3 'interface, the slash symbolizes the cleavage site in the half-strand) and Example 14071 (5'-T / GTACA-3 'interface, the slash symbolizes the cleavage site in the half-strand) 8 Genioki be amplified with overlapping detection areas in a one-pot reaction. Subsequently, unincorporated dNTPs and excess primers of the multiplex PCR are removed by means of column chromatographic purification. Alternatively, an inactivation by treatment with DNA single strand-specific 3 '- »5' exonuclease I from E. coli (EC3.1.11.1) and alkaline phosphatase (EC3.1.3.1) take place (US 2005/02148401 IAl). After this enzymatic treatment, the said enzymes are inactivated (for example 15 min 80 ^° C). The Amplifϊkatgemisch is then treated in 8 separate reaction mixtures with the 8 different restriction endonucleases, wherein at the same time or following the cleavage of the interfaces, the labeling of the selectively cleaved Amplitlkate can be done using SBE and fluorescein-ddGTP. For the genotyping of the selectively labeled DNA fragments, the mixtures are then separated in 8 separate electrophoretic runs and detected via the covalently bound fluorescein. In view of a large number of available restriction endonucleases of different recognition and cleavage specificity (Williams, 2003) and further labeling possibilities for dNTPs, ddNTPs and other chain termination reagents, a variety of combinations for the fractionated post-amplification labeling of multiplex amplification mixtures with overlapping detection regions will be apparent to those skilled in the art.

It should be noted that the method according to the invention in its analytical approach fundamentally differs from other methods for the detection of SNPs and DIPs, in which the DNA amplification method with subsequent treatment of the amplification products with corresponding enzymes is also used (eg, JR Tetr et al., 2004). Analysis of the reaction products digested by, for example, restriction endonucleases (RFLP) or other enzymes is taught in US Pat In this method, the presence or absence of the recognition sequence (s) naturally occurring in the amplified sequences is checked for the enzyme used, thereby deducing the genotype of the sequence containing, for example, an SNP, that is, the DNA to be detected Variation in this case is accompanied by an altered 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 naturally only partially present. In this case, a primer in the 5 'direction can be designed in close proximity to the DNA sequence polymorphism to be analyzed, with its sequence being altered at the 3' end by base exchange (s) of the natural sequence such that (1) enzymatic primer extension is possible and (2) at the same time a partially present in 3 'direction of the primer interface for a restriction endonuclease in the range of DNA sequence polymorphism to be analyzed is completed. Similarly US2005 / 0214840 HAI describes a method for multiplex genotyping of SNPs using restriction endonucleases, their 5 'region interface of an amplification primer was integrated. This primer is also located in the 5'-direction in the vicinity 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. Bruland and Knappskog (2004) describe the post-amplification fluorescence labeling of restriction fragments by means of SBE at naturally occurring restriction sites in order to subsequently detect sequence differences by "single strand conformation polymorphism." All of these methods are mentioned in the cited literature if they are used for multiplex applications , are carried out as one-pot reactions, ie, two or more reaction batches are not formed post-amplification and, moreover, these methods are not used for the separation of amplification mixtures with overlapping detection ranges.

In contrast, according to the invention, the specifically modified DNA amplification products which have been enzymatically, chemically and / or physically treated according to their modification are no longer available for the selected genotyping detection method, ie are not detectable, since they are eliminated from the multiplexing approach according to the invention, so that only the remaining amplification products, which are not affected by the enzymatic, chemical and / or physical treatment, are 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. Here, 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, allelspezifϊsch 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 preceded by digestion with a single strand specific DNA endonuclease selectively protected (Till et al, 2004). All other allele-specific 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 variation of this embodiment, the genotyping of all SNPs of the multiplexed approach can be done using a single color mark. For this purpose, the reaction mixture of this multiplex SBE for the detection of "n" SNPs is distributed to twice the number "2n" of reaction vessels. All further steps are analogous with the difference that only one primer, which is complementary to an allele-specifically extended SBE primer of a particular SNP locus, is used. Thus, only one allele-specific extension product is protected from digestion by a single strand-specific DNA endonuclease per approach. This arrangement allows the genotyping of SNPs from complex multiplex amplifications using optically simpler and thus particularly low-cost fluorometers. The selective cleavage of single-stranded SBE products can also be done by so-called chemical endonucleases. These are specific oligonucleotides that can form a DNA double helix by DNA-DNA hybridization with a specific target sequence. The cleavage takes place chemically by metal ion complexes, usually ethylenediaminetetraacetic acid (EDTA) iron or o-phenanthroline copper, which are covalently coupled to the oligonucleotides (Williams, 2003).

A further aspect of the present invention is a kit for carrying out the method according to the invention, wherein the kit comprises two or more primer pairs for carrying out the multiplex amplification according to the method according to the invention. The primers of the respective primer pairs each comprise DNA sequences which allow the desired target sequence (loci) to be amplified by hybridizing the primers to the DNA flanking the target sequence at a distance of about 50 to 350 base pairs. One of the two primers of the primer pair is labeled according to step a) of the method according to the invention, for example with a fluorescent dye, as described above. This labeled primer additionally contains a specific nucleic acid sequence modification as described above, with one for each locus different specific NuMeinsäuresequenzmodifikation must be present and thus with the help of two or more different enzymatic, chemical and / or physical treatment steps in two or more separate reaction mixtures each of the Loki on the enzymatic, chemical and / or physical treatment of the multiplex mixture is eliminated, and a remaining unmodified amplification product in the subsequent analysis method, as described above, clearly characterized, ie genotyped, is. Furthermore, the kit according to the invention may comprise reaction vessels into which the reaction mixture from step c) of the method according to the invention can be distributed. Furthermore, the kit according to the invention according to the multiplex method to be carried out for nucleic acid amplification suitable reagents such as buffers, nucleotides and enzymes, for. DNA polymerases, as well as suitable reference nucleic acid (s) in suitable containers.

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. For this purpose, i.a. genotyped archaeological and fossil artifacts of biological origin.

- 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 AEs ("JLoss of Heterozygosity", LOH).

The present invention is explained in more detail below using the example of combined STR and DIP analysis by means of 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 in the detection, which overlap in the detection region, as the fluorescent labels necessary for the detection detection are replaced by an amplification product Cleaved restriction endonuclease.

EXAMPLE

Multiplex PCR and genotyping of five polymorphic STR loci and a DIP locus with overlapping detection regions. For sex determination in humans, a DIP of 6 bp in the region of the amelogenin gene is suitable, which is located in one copy on both sex chromosomes in meiotic non-recombining areas (AM2, Nakahori et al., 1991). This DIP is the sequence 5'-AAAGTG-3 'which forms the base pairs 332 to 337 of the Y-specific gene copy with the accession number GenBank: M55419 and between the bases 331 and 332 of the X-specific gene copy with the accession number GenBank: M55418 is missing. To prepare a genetic fingerprint in humans, the autosomal STR markers of the C0Z> i5 'gene database standard THO1 (accession numbers: UniSTS: 156170, GenBank: NT_009237), TPOX (accession numbers: UniSTS: 240638, GenBank: NT_O3300O) were used as examples. CSFlPO (accession numbers: UniSTS: 156169; GenBank: NT_029289), D3S1358 (accession numbers: UniSTS: 148226; GenBank NT_022517) and D5S818 (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) - (S'-HEX-TTTGAATTCCCTGGGCTCTGTAAAGAA-S ': artificial sequence appendix in bold text, Underlined for EcoRI), AMR3 (SEQ ID NO: 2) (5 '

ATCAGAGCTTAAACTGGGAAGCTG-S ¹ ), D3RT1-F (SEQ ID NO: 3) (5'-6FAM-TTGATATCATGAAATCAACAGAGGCTTGC-3 ': capitalized artificial sequence appendix, site underlined for EcoRV), D3F1 (SEQ ID NO: 4) (5' -ACTGCAGTCCAATCTGGGT-3 '), D5RT3-F (SEQ ID NO: 5) (5'-6FAM-

TTTGAATTCAGCCACAGTTTACAACATTTGTATCT-3 ': boldface artificial suffix, site for EcoKi underlined), D5FT3 (SEQ ID NO: 6) (5'-GGTGATTTTCCTCTTTGGTATCC-3'), THOFT3-F (SEQ ID NO: 7) (5'-6FAM -

TTTGAATTCCTGTTCCTCCCTTATTTCCC-3 ': boldface artificial sequence attachment, site underlined for EcoRI), THOR3 (SEQ ID NO: 8) (5'-GGGAACACAGACTCCATGGTG-3'), TPFT3-H (SEQ ID NO: 9) (5'-HEX -

TTTGAATTCTTAGGGAACCCTCACTGAATG-3 ': capitalized sequence appendix in bold, site underlined for EcoRI), TPR3 (SEQ ID NO: 10) (5'-

GTCCTTGTCAGCGTTTATTTGC-3 '), CSFT1-H (SEQ ID NO: 11) (5'-HEX)

TTGATATCACAGTAACTGCCTTCATAGATAG-3 '; capitalized sequence suffix in bold, underlining interface for EcoRV) and CSRI (SΕQ ID N0: 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 prepared 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) 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, and the 25 μL volume PCR procedure consisted of 1.5 mmol / l MgCl ₂ , 200 nmol / l dNTPs. 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 μmol / l), AMRT3 (0.4 μmol / l), D3RT1-F (0.2 μmol / l) _, D3F1 (0.2 μmol / l), D5RT1-F (0.6 μmol / l) ), D5FT1 (0.6 μmol / l), THOFT3-F (0.6 μmol / l), THOR3 (0.6 μmol / l), TPFT3-H (0.2 μmol / l), TPR3 (0.2 μmol / l), CSFTl-H (0.2 μmol / l) and CSRl (0.2 μmol / l). 1). The PCR program consisted of an initial denaturation of 2 min at 94 ° C, followed by 30 cycles of 20 s at 94 ° C, 1 min at 55 ⁰ C and 30 s at 72 ⁰ C and another 60 min at 60 ⁰ 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 ⁰ C. This was followed by heat inactivation for 20 min at 82 ° C. Subsequently genioki genotyping was 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 ⁰ C for 5 min and rapidly cooled to 10 ⁰ C. The Probeniηjektion was 5 s at 15,000 V. The capillary gel electrophoresis was carried out with the polymer mixture POP-4 (Applied Biosystmens, Darmstadt, Germany) at 60 ⁰ C, 15,000 V and 20 min under denaturing conditions according to the manufacturer. 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 shown in FIG.

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Claims

claims

A method for genotyping nucleic acids, the method comprising the steps of: a) mixing an isolated sample whose nucleic acids are to be genotyped with two or more primer pairs, one primer of each of the two or more primer pairs or each between the two 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 the at least one nucleic acid modification, c) distributing 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 physis Treatment of the present in two or more reaction vessels reaction mixtures, wherein the nucleic acid-cleaving enzymes, enzyme mixtures and / or chemicals and / or the physical treatment depending on the nucleic acid sequence Modifϊkationen the amplificates are selected so that in the two or more reaction vessels present reaction mixtures in each case only one defined amplificate from a mixture of amplificates with overlapping length ranges is not cleaved by the enzymatic, chemical and / or physical treatment and the other amplificates are cleaved by the enzymatic, chemical and / or physical treatment at the nucleic acid modification, and e) detecting the uncleaved amplicons obtained from step d).

2. The method of claim 1, wherein the sample is a biological sample, 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 of microbial, plant, animal or human origin.

4. The method according to any one of the preceding claims, wherein amplifying the nucleic acids by enzyme chemical methods such as polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), ligase chain reaction (LCR), reverse transcription iigαse Chain Reaction RT (LCR), Nucleic Acid Sequence Based Amplification (NASBA), Strand Displacement Amplification (SDA), Exponential Amplification Reaction (EXPAR), Amplification Refractory Mutation System (ARMS) or ΥetrεφήmβT-AmpHfication Refractory Mutation System (Tetraprimer-ARMS).

5. The method according to any one of the preceding claims, wherein a primer of one of the two or more primer pairs and the Amplifϊkate generated by the two or more primer pairs comprises / include a detectable label, for example a fluorescent label, a colorimetrically detectable label and / or an immunologically detectable label ,

6. 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-l, 4-dichloro-6-carboxyfluorescein (NED), the colorimetrically detectable label a biotin / Strepavidin-alkaline phosphatase conjugate and the immunologically detectable label is a hapten.

7. The method according to any one of claims 5 or 6, wherein the detectable label is covalently linked to the 5 'end of the primer.

8. The method of claim 1, wherein simultaneously with or following step d) an enzymatic label is added to the cleaved nucleic acid modifications and the detection is performed in place of step e) on the basis of the labeled amplificates obtained from step d).

9. The method according to claim 8, wherein the enzymatic label in a single-base extensions (SBEs) by means of a DNA-dependent DNA polymerase and labeled Nuldeotide triphosphates or labeled chain termination reagents are made.

The method of claim 9, wherein the labeled chain-terminating reagents are 2 ', 3'-dideoxyribonucleoside triphosphates (ddNTPs).

The method of claim 9, wherein the label is a derivative of dNTPs and ddNTPs with fluorophores, biotin or radioactivity.

12. The method of claim 11, wherein the fluorophore is fluorescein-d (d) NTP, TAMRA-d (d) NTP, Cy3-d (d) NTP or Cy5-d (d) NTP.

The method of claim 11, wherein the radioactivity is αρha ³² P-dNTP, α ³² P-ddNTP, α ³³ P-dNTP or α ³³ P-ddNTP.

14. The method according to any one of the preceding claims, wherein the nucleic acid sequence modification allows enzymatic, chemical and / or physical cleavage of the amplificates and / or cleavage of the detectable label.

The method of claim 14, wherein the nucleic acid sequence modification is an endonuclease recognition and cleavage site.

The method of claim 15, wherein the endonuclease is a restriction endonuclease, an endonuclease III, a nicking endonuclease, a DNA N-glycosylase, and / or an apyridinic site (AP) DNA lyase.

17. The method of claim 14, wherein the nucleic acid sequence modification allows enzymatic generation of apyrimidine (AP) sites.

The method of claim 17, wherein the nucleic acid sequence modification is a uracyl residue which is converted by DNA uracyl N-glycosylases into an apyrimidinic (AP) site.

19. The method of claim 14, wherein the nucleic acid sequence modification allows DNA cleavage under the action of UV light.

20. The method of claim 19, wherein the nucleic acid sequence modification is a photochemically unstable base analogue such as biotin, an o-nitrophenyl and / or an o-nitrobenzyl derivative.

21. The method according to claim 1, wherein at least one nucleic acid sequence modification is in the sequence region of the template nucleic acid to be genotyped, to which the primer comprising at least one nucleic acid sequence modification hybridizes, or wherein in the region to be amplified between the nucleic acid sequence modification both primers of a primer pair is an endonuclease recognition and cleavage site, allowing for enzymatic cleavage of the amplicons and / or cleavage of the detectable label.

22. The method according to any one of the preceding claims, wherein the two or more different amplificates having an overlapping length range, two or more different nucleic acid sequence modifications which allow a selective cleavage of the amplificates and / or cleavage of the detectable label of the amplificates.

23. The method according to any one of the preceding claims, wherein the nucleic acids to be genotyped comprise at least a 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) ,

24. The method according to any one of the preceding claims, wherein the two or more amplificates generated comprise different nucleic acid sequence modifications which allow the enzymatic, chemical and / or physical cleavage of the amplificates and / or cleavage of the detectable label and the enzymatic, chemical and / or physical cleavage the amplificates and / or cleavage of the detectable label in step d) of claim 1 occur simultaneously or sequentially.

25. The method according to any one of the preceding claims, wherein prior to detecting the non-cleaved amplicon in step e) separating the amplificates by means of electrophoresis, in particular by means of denaturing capillary gel electrophoresis.

26. Use of a primer comprising a detectable label and in 3 'orientation thereof at least one nucleic acid sequence modification for carrying out a method according to any one of claims 1-7 and 14-25.

27. Use according to claim 26, wherein the detectable label comprises a fluorescent label, a colorimetrically detectable label or an immunologically detectable label.

28. Use according to claim 27, 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-fluorescem (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 Biotin / streptavidin-alkaline phosphatase conjugate and the immunologically detectable label is a hapten.

29. Use according to claim 26, wherein the nucleic acid sequence modification allows enzymatic, chemical and / or physical cleavage of the detectable label.

Use according to claim 29, wherein the primer comprises an endonuclease recognition and cleavage site.

Use according to claim 30, 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.

32. Use according to claim 29, wherein the nucleic acid sequence modification allows the enzymatic generation of apyrimidinic (AP) sites.

33. Use according to claim 32, wherein the nucleic acid sequence modification is a uracyl residue which is converted by DNA uracyl N-glycosylases into an apyrimidinic (AP) site.

34. Use according to claim 29, wherein the nucleic acid sequence modification allows DNA cleavage under the action of UV light.

35. Use according to claim 34, wherein the nucleic acid sequence modification is a photochemically unstable base analogue such as biotin, an o-nitrophenyl or an o-nitrobenzyl derivative.

36. Kit for carrying out the method according to one of claims 1-25 comprising in a suitable container two or more primer pairs, wherein in each case one primer of a primer pair has a detectable label and in 3 'orientation thereof at least one nucleic acid sequence modification.

37. The kit of claim 36, wherein the detectable label comprises a fluorescent label, a colorimetrically detectable label or an immunologically detectable label.

38. Kit according to claim 37, wherein the fluorescent label comprises 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-l, 4-dichloro-6-carboxyfluorescein (NED), the colorimetrically detectable label Biotin / streptavidin-alkaline phosphatase conjugate and the immunologically detectable label is a hapten.

39. The kit of claim 36, wherein the nucleic acid sequence modification allows enzymatic, chemical and / or physical cleavage of the detectable label.

40. The kit of claim 39, wherein the primer comprises an exploratory and cleavage site for endonucleases.

The kit of claim 40, 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.

42. The kit of claim 39, wherein said nucleic acid sequence modification allows enzymatic generation of apyrimidinic (AP) sites.

43. The kit of claim 42, wherein the nucleic acid sequence modification is a uracyl residue which is converted by DNA uracyl N-glycosylases into an apyrimidinic (AP) site.

The kit of claim 39, wherein the nucleic acid sequence modification allows DNA cleavage under the action of UV light.

45. The kit according to claim 44, wherein the nucleic acid sequence modification is a photochemically unstable base analogue such as biotin, an o-nitrophenyl or an o-nitrobenzyl derivative.