WO2007095923A1 - Procédé d'analyse de mélanges multiplex comprenant des produits d'amplification d'acides nucléiques a domaines de détection se chevauchant - Google Patents

Procédé d'analyse de mélanges multiplex comprenant des produits d'amplification d'acides nucléiques a domaines de détection se chevauchant Download PDF

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WO2007095923A1
WO2007095923A1 PCT/DE2007/000329 DE2007000329W WO2007095923A1 WO 2007095923 A1 WO2007095923 A1 WO 2007095923A1 DE 2007000329 W DE2007000329 W DE 2007000329W WO 2007095923 A1 WO2007095923 A1 WO 2007095923A1
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nucleic acid
dna
acid sequence
detectable label
cleavage
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PCT/DE2007/000329
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German (de)
English (en)
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Werner Brabetz
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Biotype Ag
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the present invention relates to the field of molecular biology nucleic acid analysis.
  • 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.
  • SNPs single nucleotide polymorphisms
  • STRs short tandem repeats
  • DIPs deletion and insertion polymorphisms
  • the invention relates to a kit with which the inventive method can be carried out.
  • 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.
  • SNPs single nucleotide polymorphisms
  • DIPs simple DNA deletions and insertions
  • repetitive DNA elements such as minisatellites, microsatellites, SBSfE, LINE, telomeres.
  • STRs Short Tandem Repeats
  • bp base pairs
  • SNPs and DIPs are usually represented by only two alleles (biallelic)
  • MI unique molecular genetic identification patterns
  • 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).
  • PCR polymerase chain reaction
  • 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.
  • 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.
  • 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 “O ⁇ igospecific Ligation Assay” (OLA).
  • SBE DNA sequence analysis
  • allele-specific DNA hybridization "eg fluorescence Resonance energy transfer probes for the real-time PCR) or "O ⁇ igospecific Ligation Assay”
  • OVA O ⁇ igospecific Ligation Assay
  • 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.
  • RNA ribonucleic acids
  • mRNA messengers RNA
  • 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).
  • STRs and DIPs 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)
  • 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)
  • dyes bind to DNA eg, ethidium bromide, Sybr Green, Sybr Gold
  • Stains All staining or silver staining silver staining
  • label-free methods eg mass spectrometry or surface plasmon resonance
  • label-free methods eg mass spectrometry or surface plasmon resonance
  • label-free methods eg mass spectrometry or surface plasmon resonance
  • label-free methods eg mass spectrometry or surface plasmon resonance
  • label-free methods eg mass spectrometry or surface plasmon resonance
  • biotin e.g., indirect colorimetric method via streptavidin and alkaline phosphatase
  • haptens e.g., in conjunction with an enzyme immunoassay
  • 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.
  • the detection unit of the analyzer used permits a clear separation of the alleles by assigning defined detection ranges.
  • fluorometers in sequencers or real-time PCR thermocylers currently have two to five color channels to distinguish appropriate fluorophores.
  • an allele-specific separation step can take place.
  • mass spectrometric and electrophoretic methods as well as addressable microspheres ("microspheres", "beads") in combination with flow cytometers or DNA chips (DNA arrays) are described.
  • 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.
  • SNPs species-specific DNA sequences or genetic polymorphisms
  • DIPs DNA-specific DNA sequences
  • STRs DNA-specific DNA sequences
  • SNPs genetic polymorphisms
  • 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.
  • 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.
  • the Amplif ⁇ kate 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.
  • 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.
  • 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.
  • 6FAM 6-carboxyfluorescein
  • HEX 4, 7, 2 ', 4', 5 ', 7'-hexachloro-6-carboxy-fluorescein
  • REN restriction endonucleases
  • 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 Ec ⁇ Bl (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).
  • PCR or “polymerase chain reaction” as used herein refer to a method for primer-based DNA amplification.
  • RT-PCR Reverse Transcriptase PCR
  • RT Polymerase chain reaction
  • NASBA Nucleic Acid Sequence Based Amplification
  • 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).
  • LCR Linker Chain Reaction
  • OLA Oligonucleotide Ligation Assay
  • JSxponential Amplification Reaction '' '' refers to an isothermal method of nucleic acid amplification.
  • 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).
  • LAMP loop-mediated isothermal amplification of DNA
  • 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).
  • helicase-dependent isothermal amplification 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).
  • single base extension 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.
  • 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)
  • DIPs deletion and insertion polymorphisms
  • DIP 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/).
  • SNP single nucleotide polymorphism
  • 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.
  • 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
  • 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
  • multiplex 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.
  • fluorophore 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-fl ⁇ orescein (TET), 2'-Chloro-5'-fluoro-7', 8 l -benzo-l, 4-dichloro-6-carboxyfluorescein (NED) (US 6,316,610), N,
  • LH 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.
  • FRET Fluorescence Energy Resonance Transfer
  • J Forster Energy Resonance Transfer 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.
  • covalently linked refers to covalent organic-chemical bonds.
  • deletions 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.
  • 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.
  • 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:
  • 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.
  • 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.
  • 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.
  • locus-specific PCR is the preferred amplification method.
  • 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.
  • 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.
  • 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.
  • suitable detector units for 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.
  • 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.
  • 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.
  • the correspondingly amplified product also contains the nucleic acid sequence modification.
  • 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').
  • 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.
  • the specific nucleic acid sequence modification may be a recognition site for jsficking endonucleases
  • the recognition sequence should be oriented so that the DNA strand is cleaved to which the detectable mark, eg the dye marking is coupled.
  • 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.
  • restriction endonucleases 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.
  • endonucleases are also known which cut PCR directly in the buffer (eg EcoKL, EcoKV, KpnT).
  • 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.
  • 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.
  • primer of the first primer pair for the amplification of STR A
  • primer of the first primer pair for the amplification of STR A
  • an EcoRV restriction site as described above may be included.
  • primer of the second primer pair for the amplification of STR B
  • primer of the third primer pair for the amplification of STR C
  • primer of the third primer pair for the amplification of STR C
  • primer of the third primer pair for the amplification of STR C
  • B. a HmdlII restriction site are inserted.
  • the inserted restriction sites are located in the 3 'direction of the fluorescent label.
  • the amplificate mixture is distributed to three reaction vessels.
  • the first reaction vessel is then subjected to restriction digestion with the restriction enzymes Ec ⁇ SCV and EcoRI.
  • 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.
  • 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.
  • STR B Only the amplificates of STR B remain intact and can be detected fluorimetrically.
  • 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.
  • 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.
  • 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).
  • restriction endonucleases are well known to those skilled in the art (see, for example, Williams (2003)).
  • 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
  • sequence-specific DNA binding domains for restriction endonucleases 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)
  • DNA cleavage for example, protein domains of
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • oligonucleotides which form specific double or triple helices with the target sequence or DNA binding proteins or corresponding protein domains mediate the sequence specificity.
  • oligonucleotides can be used which form specific double or triple helices with the primer carrying the detectable label.
  • 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.
  • metal ion complexes for example ethylenediaminetetraacetic acid (EDTA) iron or o-phenanthroline copper, which are covalently coupled to the oligonucleotides or DNA binding proteins.
  • 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.
  • 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.
  • PCR primers For genotyping according to the invention of amplification products from multiplex PCR, 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 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).
  • the specific nucleic acid sequence modifications 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.
  • dsDNA double-stranded DNA
  • 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.
  • ddNTPs 2 ', 3'-dideoxyribonucleoside triphosphates
  • suitable labels are derivatives of dNTPs and ddNTPs with fluorophores (eg fluorescein-d (eg.
  • NTP NTP
  • TAMRA-d NTP
  • Cy3-d NTP
  • Cy5-d NTP
  • biotin or radioactivity alpha 32 P-dNTP or alpha 32 P-ddNTP, alpha 33 P-dNTP or alpha 33 P-ddNTP.
  • 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 Amplif ⁇ katgemisch 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.
  • the mixtures are then separated in 8 separate electrophoretic runs and detected via the covalently bound fluorescein.
  • 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).
  • RFLP restriction endonucleases
  • 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.
  • base exchange base exchange
  • 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.
  • 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.
  • the inventive method can also be applied to single-stranded DNA products z.
  • a multiplex SBE for the detection of SNPs apply.
  • "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.
  • the reaction mixture is purified from "n” different, allelspezif ⁇ sch extended single-stranded SBE primers with streptavidin and distributed to "n” different reaction vessels.
  • the genotyping of all SNPs of the multiplexed approach can be done using a single color mark.
  • 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.
  • 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).
  • EDTA ethylenediaminetetraacetic acid
  • 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 NuMeinkladsequenzmod Enzyme 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.
  • 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.
  • kits 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.
  • 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:
  • 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.
  • 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.
  • 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 '
  • 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-
  • Genomic DNA 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 TM Human DNA Quantification Kit” (Applied Biosystems, Darmstadt, Germany).
  • the genomic DNA supplied with the kit was used as the reference DNA, and the 25 ⁇ L 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 ⁇ mol / l), AMRT3 (0.4 ⁇ mol / l), D3RT1-F (0.2 ⁇ mol / l) , D3F1 (0.2 ⁇ mol / l), D5RT1-F (0.6 ⁇ mol / l) ), D5FT1 (0.6 ⁇ mol / l), THOFT3-F (0.6 ⁇ mol / l), THOR3 (0.6 ⁇ mol / l),
  • 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 ⁇ L 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.
  • 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.
  • Electrophoresis 20 1258-1265. Lin YW, Huang MF, Chang HT (2005). Nanomaterials and chip-based nanostructures for capillary electrophoretic eparations of DNA. Electrophoresis 26: 320-330. Little MC, Andrews J, Moore R, Bustos S, Jones L, Embres C, Durmowicz G, Harris J, Berger D,
  • Genomics 9 264-269. Notomi T 5 Okayama H 5 Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hare T (2000). Loop mediated isothermal amplification of DNA. Nucleic Acids Res 28: e63. Olejnik J, Ludemann HC 5 Krzymanska-Olejnik E 5 Berkenkamp S 5 Hillenkamp F, Rothschild KJ

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Abstract

L'invention concerne le domaine de l'analyse d'acides nucléiques en biologie moléculaire. L'invention concerne en particulier un procédé de génotypage des acides nucléiques, en particulier, de génotypage d'échanges de bases individuelles ('Single Nucleotide Polymorphisms', SNPs), des microsatellites ('Short Tandem Repeats', STRs) ou des polymoprphismes de suppression et d'insertion (DIPs) dans des acides nucléiques à partir de mélanges d'amplification qui résultent de procédés multiplex d'amplification d'acides nucléiques. En outre, l'invention concerne un kit permettant la mise en oeuvre du procédé selon l'invention.
PCT/DE2007/000329 2006-02-20 2007-02-19 Procédé d'analyse de mélanges multiplex comprenant des produits d'amplification d'acides nucléiques a domaines de détection se chevauchant WO2007095923A1 (fr)

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EP3631014B1 (fr) * 2017-05-24 2022-05-11 GVG Genetic Monitoring GmbH Méthode de génotypage de souches sur souris

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011011535A3 (fr) * 2009-07-21 2011-04-14 Gen-Probe Incorporated Procédés et compositions pour la détection quantitative de séquences d'acides nucléiques sur une gamme dynamique étendue
US8628924B2 (en) 2009-07-21 2014-01-14 Gen-Probe Incorporated Methods and compositions for quantitative amplification and detection over a wide dynamic range
JP2014030431A (ja) * 2009-07-21 2014-02-20 Gen Probe Inc 拡張されたダイナミックレンジにわたる核酸配列の定量的検出のための方法および組成物
US8932817B2 (en) 2009-07-21 2015-01-13 Gen-Probe Incorporated Methods for quantitative amplification and detection over a wide dynamic range
EP3018218A1 (fr) * 2009-07-21 2016-05-11 Gen-Probe Incorporated Procédés et compositions pour la détection quantitative de séquences d'acides nucléiques sur une plage dynamique étendue
US9347098B2 (en) 2009-07-21 2016-05-24 Gen-Probe Incorporated Reaction mixtures for quantitative amplification and detection over a wide dynamic range
US9856527B2 (en) 2009-07-21 2018-01-02 Gen-Probe Incorporated Methods for quantitative amplification and detection over a wide dynamic range
EP3330385A1 (fr) * 2009-07-21 2018-06-06 Gen-Probe Incorporated Procédés et compositions pour la détection quantitative de séquences d'acides nucléiques sur une plage dynamique étendue
EP3631014B1 (fr) * 2017-05-24 2022-05-11 GVG Genetic Monitoring GmbH Méthode de génotypage de souches sur souris

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