RU2558231C2 - Method, test-system and primers for determination of haplogroups of human y-chromosome - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: inventions relate to the field of DNA-genealogy and deal with a method of determining haplogroups of human Y-chromosomes, a test-system and oligonucleotide primers. The characterised method is realised in two stages. At the first stage genetic typing is carried out by basic haplogroups R,N,J,I,Q,C,E,D,G,O of an Y-chromosome tree by multiplex PCR with the application of specific oligonucleotide primers of the first and second sets of the test-system. At the second stage, if mutation is detected, additional multiplex PCR for typing by subhaplogroups is carried out. If at the first stage mutation is not detected, multiplex PCR for typing by rare haplogroups A,C3,F,H,K,L,O3 and T is carried out. The area of annealing primers outside mutation zones within the limits of species specificity corresponds to sequences from the first set of the test-system. The area of annealing of the oligonucleotide primers, directly adjoining the mutation zone within the limits of species specificity corresponds to sequences from the second set of the test-system.

EFFECT: inventions can be applied for the determination of haplogroups of the human Y-chromosome.

3 cl, 2 dwg, 7 tbl, 2 ex

Description

The invention relates to the field of DNA genealogy, molecular biology and biotechnology and can be used to establish haplogroups of the Y chromosome and ethnicity of a person. The invention will be of interest to ethnographers, anthropologists, population geneticists, people interested in the history of their ancestors or searching for their relatives among namesakes, as well as in forensics.

In DNA genealogy, in order to determine the haplogroup of the Y chromosome, approximately 300 SNP polymorphisms must be sequentially typed, because having typed only one SNP polymorphism, the researcher will not be able to determine which ethnicity a person belongs to, which is associated with the sequential accumulation of mutations on the Y chromosome. Traditionally, for the genotyping of single nucleotide polymorphisms (SNPs) of the Y chromosome, a hierarchical algorithm is used, according to the established sequence of accumulation of mutations on the Y chromosome [Cinnioglu C, King R., Kivisild T. et al. Excavating Y-chromosome haplotype strata in Anatolia // Hum. Genet. - 2004 - V. 114. - P. 127-148 .; Cruciani F., Santolamazza P., Shen P., et al. A Back Migration from Asia to Sub-Saharan Africa Is Supported by High-Resolution Analysis of Human Y-Chromosome Haplotypes // Am. J. Hum. Genet. - 2002. - V. 70. - P. 1197-1214 .; Hammer M.F. A recent common ancestry for human Y-chromosomes // Nature. - 1995a. - V. 378. - P. 376-378 .; Karafet T. M., Mendez F. L., Meilerman M. B., et al. New binary polymorphisms reshape and increase resolution of the human Y chromosomal haplogroup tree. // Genome Res. 2008 18: 830-838 originally published online April 2, 2008. - P. 830-838 .; Karafet T.M., Osipova L.P., Gubina M.A. et al. High Levels of Y-chromosome differentiation among Native Siberian Populations and the Genetic Signature of a Doreal-Galherer Way of Life // Human Biology. - 2002. - V. 74. - No. 6. - P. 761-789 .; Underhill P. A., Passarino G., Lin A. A. et al. The phylogeography of Y chromosome binary haplotypes and the origins of modern human populations // Ann. Hum. Genet. - 2001. - V. 65. P. 43-62 .; Underhill P.A., Shen P., Lin A.A. et al. Y chromosome sequence variation and the history of human populations // Nat. Genet. - 2000. - V. 26. - 358-361]. However, this algorithm is very time-consuming and more suitable for routine use in research laboratories and completely unsuitable for commercial DNA genealogy. Currently, methods for typing single nucleotide polymorphisms such as RFLP analysis and TaqMan method are known for determining Y-chromosome haplogroups [ Brown T.A. Genomes. - M. - Izhevsk: Institute for Computer Research. - 2011 .-- 944 p .; Rebrikov D.V. and other real-time PCR. - M .: BINOM. Laboratory of Knowledge, 2009 .-- 223 p., Luo Ziyi, Wu Yumei. Real-time fluorescence quantification PCR (Polymerase Chain Reaction) kit and method for detecting Y-chromosome micro-deletion. CN102876776 (A) 2013-01-16.]. The essence of RFLP analysis is as follows: the enzyme, restriction endonuclease, binds to a DNA molecule in the region of a specific sequence and makes a double-stranded incision in this sequence or near it. After DNA treatment with restriction endonucleases, the obtained fragments can be examined by agarose gel electrophoresis [Brown T.A. Genomes. - M. - Izhevsk: Institute for Computer Research. - 2011. - 944 s]. The essence of the TaqMan method is that in this approach, an oligonucleotide complementary to the PCR product is labeled with a fluorophore and a fluorescence quencher. In the absence of a target, the fluorophore and quencher are brought together and fluorescence is suppressed. Upon accumulation of the corresponding reaction product, the sample hybridizes to an amplicon, which leads to its destruction due to the 5′-endonuclease activity of Taq polymerase. The signal intensity increases with each PCR cycle in proportion to the accumulation of amplicons [D. Rebrikov and other real-time PCR. - M .: BINOM. Laboratory of Knowledge, 2009. - 223 p.].

Significant disadvantages of the above methods is that only one single nucleotide polymorphism (SNP) can be determined per reaction, which makes these methods expensive, requiring a significant investment of time and human labor.

Known methods based on chip technologies of foreign production, which allow you to type a large number of single-nucleotide polymorphisms at the same time. The essence of the method is that oligonucleotides are synthesized in situ on the surface of a plate of glass, silicon or polymer, resulting in a DNA chip. In this case, it is possible to achieve a density of up to 300,000 oligonucleotides per cm2, so that when using DNA chips to typify SNPs, 150,000 polymorphisms can be typed in one experiment (provided that there are oligonucleotides for both alleles of each SNP [Brown T. A. Genomes. - M. - Izhevsk: Institute for Computer Research. - 2011. - 944 p.] However, these technologies for determining ethnicity are very expensive, require appropriate technology platforms for chip analysis, consumables are very expensive and, accordingly -retarded, road services provided by these technologies.

The known method according to US patent No. US 20030225530 A1, publ. 12/04/2003, intended for forensic medicine and personality identification, which allows obtaining additional information about the ethnic characteristics of a person using a DNA sample, the essence of which is to use a database containing information about the presence and / or identity of one or more variations in one or more DNA loci (HUMVWFA31, HUMTH01, HUMFIBRA, D8S1179, D21S11, D18S51, D3S1358, D2S1338, D16S539 or D19S433) for a set of reference samples with a known physical characteristic, forming groups that share common characteristics in frequency a specific locus.

The disadvantage of this method is that its use does not allow to determine the ethnicity of a person by the haplogroup of the Y chromosome.

A known method for determining the detection of microdeletions of the Y chromosome [Luo Ziyi, Wu Yumei. Real-time fluorescence quantification PCR (Polymerase Chain Reaction) kit and method for detecting Y-chromosome micro-deletion. CN102876776 (A) 2013-01-16]. The invention is realized using a fluorescence quantitative polymerase chain reaction in real time by the detection of Y-chromosome microdeletions, in which PCR is based on the fact that the primer probe is selected to detect a specific locus: primer probe combination for locus detection SY84, primer probe combination for locus detection SY127, primer probe combination for locus detection SY134, primer probe combination for locus detection SY254, primer probe combination for locus detection SY255. Microdilations at the Y chromosome loci can be detected by a combination of real-time fluorescence quantitative PCR and real-time Taqman probes using real-time fluorescence quantitative PCR. The fluorescent signals of each PCR cycle increase in proportion to the accumulation of the product.

The main disadvantage of this method is the length of the determination of polymorphisms of the Y chromosome, since sequential PCR reaction with each primer is necessary to determine each SNP locus.

This disadvantage is deprived of a method for determining the pure breed of animals - agricultural objects, adopted as a prototype, according to the patent of the Russian Federation No. 2477607, publ. 03/20/2013, including the extraction of DNA from biological material, conducting a multiplex PCR reaction of 13 microsatellite loci (TGLA126, ILSTS005, ETN185, TGLA122, INRA023, ILSTS006, TGLA227, ETN225, ETN10, BM1818, BM-1111, SPS test, SPS ,24, SPS DNA examination of animals and analysis of the resulting products on a DNA analyzer.

The disadvantage of this technical solution is that it is intended for DNA examination of animals and cannot be used to determine the haplogroup of the Y chromosome and ethnicity of a person using a DNA sample.

Currently, the express method is not known from the prior art based on the multiplex determination of haplogroups of the Y chromosome and the establishment of a person’s ethnicity using a test system that could be used in DNA genealogy studies, so the introduction of such a method on the market seems very relevant.

Known test system "Set for the detection of microdeletions of the Y-chromosome", which includes: 1) reagents for the PCR reaction; 2) primers for amplification of the SY84 sequence of the AZFa region of the Y chromosome; primers for amplification of the sequence of the SY86 region of the AZFa region of the Y-chromosome region; primers for amplification of the sequence of the SY127 AZFb region of the Y chromosome; primers for amplification of the sequence of the SY134 AZFb region of the Y chromosome; primers for amplification of the sequence of the SY254 AZFc region of the Y chromosome; primers for amplification of the sequence of SY255 AZFc region of the Y chromosome, and primers for amplification of the SRY gene described in the source [Kun Song; Jiancheng Xu; Shuyi Wang; Cuilian Sun. Nanchang adicon clinical tersting inst with ltd. Kit for detecting microdeletion of Y chromosome CN102796819 (A) 2012-11-28.]. This test system, selected for the prototype of the proposed test system, provides for a sequential PCR reaction with each primer. The disadvantage is the impossibility of conducting a multiplex analysis, which determines the duration of the determination of polymorphisms of the human Y-chromosome.

Known primer sequences for determining the polymophism of the human Y chromosome are given in the publications [Cinnioglu C., King R., Kivisild T. et al. Excavating Y-chromosome haplotype strata in Anatolia // Hum. Genet. - 2004. - V.114. - P.127-148; Underhill P. A., Passarino G., Lin A. A. et al. The phylogeography of Y chromosome binary haplotypes and the origins of modern human populations // Ann. Hum. Genet. - 2001. - V.65. P.43-62]. However, these primer sequences (Table 1) do not meet the requirements for multiplex analysis of SNP polymorphisms of the Y chromosome, because have different physical characteristics.

The objective of the proposed group of inventions is the creation of a method, test system and primers for determining haplogroups of the Y chromosome and ethnicity of a person, which by multiplex analysis will allow to simultaneously determine several single nucleotide polymorphisms of the human Y chromosome.

The technical result of the proposed group of inventions is that when genotyping SNP polymorphisms of a human Y chromosome by multiplex analysis in two stages, about 20 SNP markers for one DNA sample can be directly typed, i.e. quickly, absolutely accurately and reliably determine the haplogroup, subhaplogroup and ethnicity of a person using a DNA sample.

To achieve this goal, a method is proposed that includes the extraction of DNA from biological material, conducting a multiplex PCR reaction using a test system, analysis of the resulting products on a DNA analyzer, which introduced the following new features: the method is carried out in two stages using a test system, containing two sets: at the first stage, genotyping is performed by multiplex PCR reaction using multiplexes of 20 specific oligonucleotide primers for genotyping for the main haplogroups N, J, I, Q, C, E, D, G, O of the Y chromosome tree from the first set of the test system, then the PCR amplification on the columns is purified and SNaPshot reaction is performed using multiplexes of 10- specific oligonucleotide primers for genotyping according to the main haplogroups from the second set of the test system, then at the second stage according to the analysis results, if the mutation is detected at the first stage, genotyping is carried out by multiplex PCR reaction by subhaplogroups using multiplexes of 62 specific oligonucleotide primers for genotyping by subgaplogroups from the first set of the test system and then, after purification of the amplification using columns, the SNaPshot reaction is carried out by multiplex analysis using multiplexes of 28 specific oligonucleotide primers for genotyping by subgaplogroups from the second set of test system, or, if no mutation is detected at the first stage, genotyping is performed for rare haplogroups: A, C3, F, H, K, L, O3, T using multiplexes of 16 specific oligonuclei of leotide primers for genotyping by rare haplogroups from the first set of the test system, after purification of the amplification using columns, the SNaPshot reaction is carried out by multiplex analysis using multiplexes of 8 specific oligonucleotide primers for genotyping by rare haplogroups from the second set of test system, and the products are purified by enzyme TSAP, after which they are analyzed.

As a result, both in the presence of a mutation and without it, in two stages about 20 SNP markers for one DNA sample will be genotyped immediately and a subhaplogroup will be determined with 100% accuracy, which significantly reduces the cost and speeds up the study itself. There is no need to sequentially genotype a DNA sample for all known SNP markers of the Y chromosome, of which there are about 300, in order to establish the subhaplogroup and ethnicity of a person.

In FIG. 1 presents a scheme for genotyping SNPs of the Y chromosome according to the proposed method.

In FIG. 2 presents a scheme for determining sub-haplogroups of the Y chromosome, indicating the corresponding markers according to the proposed method.

To implement the claimed method, a test system is proposed that contains a kit that contains a mixture for the PCR reaction and primer sets for amplification of the DNA sequence, which contains the following new features: the test system additionally includes a second kit for the SNaPshot reaction, The first set includes:

1. The mixture for the PCR reaction is 1.5 ml.

2. Multiplex sets of specific oligonucleotide primers of 1.5 ml each.

2.1. Multiplexes of 20 specific oligonucleotide primers for genotyping according to the main haplogroups R, N, J, I, Q, C, E, D, G, O of the Y chromosome tree, SNP markers: M207, M231, M304, M170, M242, M216, M96 , M174, M201, P186, the sequences of which and their corresponding SNP markers are presented in Table 2;

2.2. Multiplexes of 62 specific oligonucleotide primers for genotyping by subhaplogroups, the sequences of which and their corresponding SNP markers are presented in Table 3;

2.3. Multiplexes of 10 specific oligonucleotide primers for the genotyping of rare haplogroups - A, C3, F, H, K, L, O3, T, the sequences of which and their corresponding SNP markers are presented in Table 4.

The second kit for conducting SNaPshot reactions contains multiplex sets of specific oligonucleotide primers of 1.5 ml each:

1. Multiplexes of 10 specific oligonucleotide primers for genotyping of the main haplogroups of the Y chromosome, the sequences of which and their corresponding SNP markers are presented in Table 5;

2. Multiplexes of 28 specific oligonucleotide primers for genotyping by subhaplogroups, the sequences of which and their corresponding SNP markers are presented in Table 6;

3. Multiplexes of 8 specific oligonucleotide primers for genotyping of rare haplogroups, the sequences of which and their corresponding SNP markers are presented in Table 7.

To implement the method for determining haplogroups of the Y chromosome and ethnicity of a person, specific oligonucleotide primers have been developed that are used as multiplex sets of a test system for amplification of areas of the non-recombining part of the Y chromosome.

The design of specific oligonucleotide primers (tables 2, 3 and 4) for the first set was carried out on the basis of the following principles: the annealing region of the oligonucleotide primers should be outside the mutation zones within species specificity in the range of 55-64 ° C; GC composition in the range of 40-60%; in the sequence of the primer there should be no stable secondary structures - hairpins and dimers.

The design of specific oligonucleotide primers (tables 5, 6 and 7) for the second set was carried out on the basis of the following principles: the annealing region of oligonucleotide primers should be directly adjacent to the mutation zone within species specificity in the range of 50 ° C ± 2 ° C; in the sequence of the primer there should be no stable secondary structures - hairpins and dimers; to the 5 ′ ends of each primer, GACT tails must be attached in accordance with the given length of the oligonucleotide primer.

To exclude amplification of non-target loci, the uniqueness of each primer was confirmed by a search on the database of sequenced genomes http://www.ensembl.org.

The claimed method based on the proposed test system containing the developed specific oligonucleotide primers is as follows.

DNA is isolated from any biological material (buccal epithelium, hair, nails or blood) by any method suitable for isolating DNA from a particular type of biomaterial.

At the first stage, the extracted DNA is subjected to a multiplex polymerase chain reaction for the synthesis of SNP loci of the Y chromosome for genogenotyping in the main haplogroups - R, N, J, I, Q, C, E, D, G, O of the Y chromosome tree using multiplexes 20 specific oligonucleotide primer mixtures from the first set of test systems (table 2). The amplification obtained by PCR is subjected to purification using columns. Next, the SNaPshot reaction is carried out using multiplexes of 10 specific oligonucleotide primers (Table 5) from the second set of the test system, the products of the SNaPshot reaction are purified by the TSAP enzyme, and the DNA analyzer is analyzed by capillary gel electrophoresis. At the second stage, if a mutation is detected at the first stage, genotyping by subhaplogroups is performed using multiplexes of 62 specific oligonucleotide primers (Table 3) for genotyping by subhaplogroups from the first set of the test system; after purification of the amplification using columns, the SNaPshot reaction is performed by multiplex analysis using multiplexes of specific oligonucleotide primers (Table 6) for typing by subhaplogroups from the second set of the test system, and if at the first stage the mutation is not It has been shown that genotyping is performed for rare haplogroups: A, C3, F, H, K, L, O3, T using multiplexes of specific oligonucleotide primers (Table 4) for typing on rare haplogroups from the first set of the test system, after purification of the amplification using columns carry out SNaPshot reactions using multiplexes of specific oligonucleotide primers (Table 7) for typing on rare haplogroups from the second set of test systems, the products formed in the second stage are purified with TSAP enzyme, and then DNA analysis is performed on an analyzer by capillary gel electrophoresis.

The polymerase chain reaction (PCR) of the synthesis of SNP loci of the Y chromosome is performed in a reaction mixture of 25 μl, 67 mM Tris-HCl (pH = 8.8), 2.5 mM MgCl2, 1 ng of genomic DNA, 10 pM of each primer, 200 μM dATP, dGTP, dCTP, dTTP and 1 unit of active Taq polymerase. The PCR regimen is as follows: 1) denaturation 94 ° C - 3 min. 30 sec .; 40 cycles: 2) denaturation 94 ° С - 30 sec .; primer annealing: 55 ° С-64 ° С - 30 sec .; elongation 72 ° C - 1 min .; 3) final elongation 72 ° C -10 min.

SNaPshot reaction is carried out according to the protocol of the manufacturer: reaction mixture - 5 μl .; specific primers - 1 μl .; deionized water - 1 μl; the amplification is 3 μl. according to the following regime: 25 cycles 1) denaturation 96 ° С - 10 sec .; 2) annealing of primers: 50 ° С - 5 sec .; 3) elongation 60 ° C - 30 sec.

To purify the SNaPshot reaction product with TSAP: 1 μl is added. TSAP enzyme; purification scheme: 37 ° С - 60 min .; 75 ° C - 15 min. Then, detection is carried out on a DNA analyzer by capillary gel electrophoresis.

Examples of the implementation of the group of claimed inventions.

one). Citizen M. took a sample of biological material - buccal epithelium. DNA was isolated by the standard method. Next, a PCR reaction was carried out according to the following protocol: deionized water - 12.8; buffer - 2.5; MgCl ₂ - 2.5 μl; multiplexes of specific oligonucleotide primers for genotyping according to the main haplogroups (Table 2) from the first set of the test system — 5.5 μl .; dNTP - 0.2 μl .; Taq polymerase - 0.5 μl .; DNA - 1 μl. PCR mode: 1) denaturation 94 ° C - 3 min. 30 sec .; 40 cycles: 2) denaturation 94 ° С - 30 sec .; primer annealing: 64 ° С - 30 sec .; elongation 72 ° C - 1 min .; 3) final elongation 72 ° C - 10 min. The amplification was purified on columns according to the manufacturer's instructions. Next, the SNaPshot reaction was carried out according to the manufacturer's protocol: reaction mixture - 5 μl; multiplexes of specific oligonucleotide primers (Table 5) for genotyping according to the main haplogroups from the second set of the test system - 1 μl; deionized water - 1 μl; the amplification is 3 μl. according to the following regime: 25 cycles 1) denaturation 96 ° С - 10 sec .; 2) annealing of primers: 50 ° С - 5 sec .; 3) elongation 60 ° C - 30 sec. Purification of the SNaPshot product with TSAP enzyme by adding 1 μl. TSAP enzyme; purification scheme: 37 ° С - 60 min .; 75 ° C - 15 min. The analysis was performed on a DNA analyzer by capillary gel electrophoresis according to the following protocol of the manufacturer: Hi-Di formamide - 9 μl .; LIZ120 - 0.5 μl .; the product is 0.5 μl. The result was evaluated according to the following criteria: allele A - green signal; allele T - red signal; G allele - blue signal; allele C is a black signal. Each locus must correspond to one of the following options:

M96 - C → A (38-41 bp) haplogroup E

M231 - G → A (42-45 bp) haplogroup N

M216 - G → A (48-51 bp) haplogroup C

M170 - A → G (54-57 bp) haplogroup I

M174 - T → C (60-63 bp) haplogroup D

M201 - C → A (66-69 bp) haplogroup G

M207 - A → G (72-75 bp) haplogroup R

M304 - A → (78-81 bp) haplogroup J

P186 - C → A (90-93 bp) haplogroup O

M242 - G → A (96-99 bp) haplogroup Q

The following result is obtained:

M96 - C (38-41 bp)

M231 - G (42-45 bp)

M216 - G (48-51 bp)

M170 - A (54-57 bp)

M174 - T (60-63 bp)

M201 - C (66-69 bp)

M207 - G (72-75 bp)

M304 - A (78-81 bp)

P186 - C (90-93 bp)

M242 - G (96-99 bp)

The analysis result for the main haplogroups showed the identification of the European haplogroup R, therefore, at the second stage of determining the subhaplogroup, specific primers were used (Table 3) for genotyping by subhaplogroups from the first set of the test system.

The PCR reaction in the second stage was carried out according to the following protocol: deionized water - 12.8; buffer - 2.5; MgCl ₂ - 2.5 μl; multiplexes of specific oligonucleotide primers (Table 3) for genotyping by subhaplogroups - 5.5 μl; dNTP - 0.2 μl; Taq polymerase - 0.5 μl; DNA - 1 μl. PCR mode: 1) denaturation 94 ° C - 3 min. 30 sec .; 40 cycles: 2) denaturation 94 ° С - 30 sec .; primer annealing: 64 ° С - 30 sec .; elongation 72 ° C - 1 min .; 3) final elongation 72 ° C - 10 min. The amplification was purified on columns according to the manufacturer's instructions. Next, the SNaPshot reaction was carried out according to the manufacturer's protocol: reaction mixture - 5 μl; multiplexes of specific oligonucleotide primers (Table 6) for genotyping by subhaplogroups from the second set of the test system - 1 μl; deionized water - 1 μl; amplification - 3 μl, according to the following regime: 25 cycles 1) denaturation 96 ° С - 10 sec .; 2) annealing of primers: 50 ° С - 5 sec .; 3) elongation 60 ° C - 30 sec. Purification of the SNaPshot product with TSAP enzyme by adding 1 μl. TSAP enzyme according to the scheme of purification: 37 ° С - 60 min .; 75 ° C - 15 min. The analysis was performed on a DNA analyzer by capillary gel electrophoresis according to the following protocol of the manufacturer: Hi-Di formamide - 9 μl .; LIZ120 - 0.5 μl .; the product is 0.5 μl. The result was evaluated according to the following criteria: allele A - green signal; allele T - red signal; G allele - blue signal; allele C is a black signal. Each locus must correspond to one of the following options:

M173 - T → C (80-83 bp) haplogroup R1

M343 - A → C (56-59 bp) haplogroup R1b

M17 - del G (44-47 bp) - when del G is detected, the T allele corresponding to haplogroup R1a1 will be detected, the absence of del G corresponds to the absence of a mutation

M417 - G → A (68-71 bp) haplogroup R1a1a1

M73 - del AC (38-41 bp) haplogroup R1b1b1

M269 - T → C (50-53 bp) haplogroup R1b1b2

M124 - G → A (62-65 bp) haplogroup R2

The following result is obtained:

M173 - C (80-83 bp) haplogroup R1

M343 - A (56-59 bp) haplogroup R1b

M17 - del G (44-47 bp) - T allele detected

M417 - G (68-71 bp) haplogroup R1a1a1

M73 - nondel AC (38-41 bp) haplogroup R1b1b1

M269 - T (50-53 bp) haplogroup R1b1b2

M124 - G (62-65 bp) haplogroup R2

The result of the analysis by subhaplogroups revealed a subhaplogroup R1a1.

Consequently, the ethnicity of citizen M. is European, and more specifically, Eastern European.

2). Citizen N. took a sample of biological material - buccal epithelium. DNA was isolated by the standard method. At the first stage, a PCR reaction was carried out according to the following protocol: deionized water - 12.8; buffer - 2.5; MgCl2 - 2.5 μl; multiplexes of specific oligonucleotide primers (Table 2) for genotyping according to the main haplogroups from the first set of the test system — 5.5 μl .; dNTP - 0.2 μl .; Taq polymerase - 0.5 μl .; DNA - 1 μl. PCR mode: 1) denaturation 94 ° C - 3 min. 30 sec .; 40 cycles: 2) denaturation 94 ° С - 30 sec .; primer annealing: 64 ° С - 30 sec .; elongation 72 ° C - 1 min .; 3) final elongation 72 ° C - 10 min. The amplification was purified on columns according to the manufacturer's instructions. Next, the SNaPshot reaction was carried out according to the manufacturer's protocol: reaction mixture - 5 μl; multiplexes of specific oligonucleotide primers (Table 5) for genotyping according to the main haplogroups from the second set of the test system - 1 μl; deionized water - 1 μl; the amplification is 3 μl. according to the following regime: 25 cycles 1) denaturation 96 ° С - 10 sec .; 2) annealing of primers: 50 ° С - 5 sec .; 3) elongation 60 ° C - 30 sec. Purification of the SNaPshot product with TSAP enzyme by adding 1 μl. TSAP enzyme .; purification scheme: 37 ° С - 60 min .; 75 ° C - 15 min. The analysis was performed on a DNA analyzer by capillary gel electrophoresis according to the following protocol of the manufacturer: Hi-Di formamide - 9 μl; LIZ120 - 0.5 μl .; the product is 0.5 μl. The result was evaluated according to the following criteria: allele A - green signal; allele T - red signal; G allele - blue signal; allele C is a black signal. Each locus must correspond to one of the following options:

M96 - C → A (38-41 bp) haplogroup E

M231 - G → A (42-45 bp) haplogroup N

M216 - G → A (48-51 bp) haplogroup C

M170 - A → G (54-57 bp) haplogroup I

M174 - T → C (60-63 bp) haplogroup D

M201 - C → A (66-69 bp) haplogroup G

M207 - A → G (72-75 bp) haplogroup R

M304 - A → C (78-81 bp) haplogroup J

P186 - C → A (90-93 bp) haplogroup O

M242 - G → A (96-99 bp) haplogroup Q

The following result is obtained:

M96 - C (38-41 bp)

M231 - G (42-45 bp)

M216 - G (48-51 bp)

M170 - A (54-57 bp)

M174 - T (60-63 bp)

M201 - C (66-69 bp)

M207 - A (72-75 bp)

M304 - A (78-81 bp)

P186 - C (90-93 bp)

M242 - G (96-99 bp)

The analysis result for the main haplogroups showed the absence of mutations, not a single haplogroup was identified, therefore, at the second stage, multiplexes of specific oligonucleotide primers were used (Table 4) for genotyping by rare haplogroups from the first set of the test system.

At the second stage, to determine rare haplogroups, a PCR reaction was performed to determine rare haplogroups according to the following protocol: deionized water - 12.8; buffer - 2.5; MgCl2 - 2.5 μl; multiplexes of specific oligonucleotide primers (Table 4) for genotyping by rare haplogroups - 5.5 μl .; dNTP - 0.2 μl; Taq polymerase - 0.5 μl; DNA - 1 μl. PCR mode: 1) denaturation 94 ° C - 3 min. 30 sec .; 40 cycles: 2) denaturation 94 ° С - 30 sec .; primer annealing: 64 ° С - 30 sec .; elongation 72 ° C - 1 min .; 3) final elongation 72 ° C - 10 min. The amplification was purified on columns according to the manufacturer's instructions. Next, the SNaPshot reaction was carried out according to the manufacturer's protocol: reaction mixture - 5 μl; multiplexes of specific oligonucleotide primers (Table 7) for genotyping by rare haplogroups - 1 μl; deionized water - 1 μl; amplification - 3 μl according to the following regime: 25 cycles 1) denaturation 96 ° С - 10 sec .; 2) annealing of primers: 50 ° С - 5 sec .; 3) elongation 60 ° C - 30 sec. Purified SNaPshot of the product with TSAP enzyme by adding 1 μl of TSAP enzyme; purification scheme: 37 ° С - 60 min .; 75 ° C - 15 min. The analysis was performed on a DNA analyzer by capillary gel electrophoresis according to the following protocol of the manufacturer: Hi-Di formamide - 9 μl .; LIZ120 - 0.5 μl; product - 0.5 μl. The result was evaluated according to the following criteria: allele A - green signal; allele T - red signal; G allele - blue signal; allele C is a black signal. Each locus must correspond to one of the following options:

M91 - A → G (50-54 bp) haplogroup A

M217 - A → C (68-70 bp) haplogroup C3

M89 - C → T (56-59 bp) haplogroup F

M69 - A → G (62-65 bp) haplogroup H

M9 - C → G (84-87 bp) haplogroup K

M11 - A → G (38-42 bp) haplogroup L

M122 - A → G (50-54 bp) haplogroup O3

M70 - T → G (44-47 bp) haplogroup T

The following result is obtained:

M91 - A (50-54 bp) haplogroup A

M217 - A (68-70 bp) haplogroup C3

M89 - C (56-59 bp) haplogroup F

M69 - A (62-65 bp) haplogroup H

M9 - G (84-87 bp) haplogroup K

M11 - G (38-42 bp) haplogroup L

M122 - A (50-54 bp) Haplogroup O3

M70 - T (44-47 bp) haplogroup T

The analysis of rare haplogroups revealed haplogroup L, therefore, the ethnicity of citizen N is South Asian.

Thus, the above examples confirm that the claimed method, which is carried out by the proposed test system containing the developed specific oligonucleotide primers, allows genotyping about 20 SNP markers for one DNA sample in just two steps and determining the human haplogroup and ethnicity with 100% accuracy , which significantly reduces the cost and speeds up the study itself.

Bibliography

1. Bosch E., Calafell F., Comas D. et al. High-resolution analysis of human Y-chromosome variation shows a sharp discontinuity and limited gene flow between northwestern Africa and the Iberian Peninsula // Am. J. Hum. Genet. - 2001. - V. 68. - P. 1019-1029.

2. Cruciani F., La Fratta R., Santolamazza P. et al. Phylogeographic Analysis of Haplogroup EZ (E-M215) Y Chromosomes Reveals Multiple Migratory Events Within and Out Of Africa // Am. J. Hum. Genet. - 2004. - V. 74. - P. 1014-1022.

3. Cruciani F., La Fratta R., Torroni A. et al. Molecular Dissection of the Y Chromosome Haplogroup E-M78 (E3bla): A Posteriori Evaluation of a Microsatellite-Network-Based Approach Through Six New Biallelic Markers // Human mutation Mutation in Brief. - 2006. - P. 1-10.

4. Di Giacomo F., Luca F., Popa L. O. et al. Y chromosomal haplogroup J as a signature of the post-neolithic colonization of Europe // Hum. Genet. - 2004. - V. 115. - P. 357-371.

5. Hammer M.F., Karafet T., Rasanayagam A. et al. Out of Africa and back again: nested cladistic analysis of human Y chromosome variation // Mol. Biol. Evol. - 1998. - V. 15. - P. 427-441.

6. Hammer M. F., Chamberlain V. F, Kearney V. F. et al. Population structure of Y chromosome SNP haplogroups in the United States and forensic implications for constructing Y chromosome STR databases // Forensic Science International. - 2005 .-- P. 1-11.

7. Karafet T. M., Zegura S. L., Posukh O. et al. Ancestral Asian source of New World Y-chromosome founder haplotypes // Am. J. Hum. Genet. - 1999. - V. 64. - P.817-831.

8. Karafet T.M., Mendez F. L., Meilerman M. B., et al. New binary polymorphisms reshape and increase resolution of the human Y chromosomal haplogroup tree // Genome Res. - 2008 .-- P. 830-838.

9. Rootsi S, Magri C, Kivisild T. et. al. Phylogeography of Y-Chromosome Haplogroup I Reveals Distinct Domains of Prehistoric Gene Flow in Europe // Am. J. Hum. Genet. - 2004. - V. 75. - P. 128-137.

10. Rootsi S., Zhivotovsky L.A., Baldovic M. et al. A counter-clockwise northern route of the Y-chromosome haplogroup N from Southeast Asia towards Europe // European Journal of Human Genetics. - 2006. - P. 1-8.

11. Semino O., Passarino G., Brega A. et al. A view of the Neolithic demic diffusion in Europe through two Y chromosome-specific markers // Am. J. Hum. Genet. - 1996 - V. 59. - P. 964-968.

12. Semino O., Passarino G., Oefner P. J. et al. The Genetic Legacy of Paleolithic Homo sapiens sapiens in Extant Europeans: A Y Chromosome Perspective // Science. - 2000. - V. 290. - P. 1155-1159.

13. The Y Chromosome Consortium 1. A Nomenclature System for the Tree of Human Y-Chromosomal Binary Haplogroups // Genome Research. - 2002. - V. 12. - P. 339-348.

14. Underhill P.A., Jin, L. Zemans R. et al. A pre-Columbian Y chromosome-specific transition and its implications for human evolutionary history // Proc. Natl. Acad. Sci. - 1996. - V. 93. - P. 196-200.

15. Underhill P.A., Jin L., Lin A. A. et al. Detection of numerous Y chromosome biallelic polymorphisms by denaturing high-performance liquid chromatography // Genome Res. - 1997. - V. 7. - P. 996-1005.

16. Underhill P.A., Shen P., Lin A.A. et al. Y chromosome sequence variation and the history of human populations // Nat. Genet. - 2000. - V. 26. - 358-361.

17. Underhill P. A., Passarino G., Lin A. A. et al. The phylogeography of Y chromosome binary haplotypes and the origins of modern human populations // Ann. Hum. Genet. - 2001. - V. 65. P. 43-62.

18. Wells R. S., Yuldasheva N., R. Ruzibakiev et al. The Eurasian Heartland: A continental perspective on Y-chromosome diversity // PNAS. - 2001. - V. 98, No. 18. - P. 10244-10249.

19. Whit Athey T. and Nordtvedt K. Resolving the Placement of Haplogroup I-M223 in the YChromosome Phylogenetic Tree // Journal of Genetic Genealogy. - 2005 .-- P. 54-55.

20. Zegura S.L., Karafet T.M., Zhivotovsky L.A. et al. High-Resolution SNPs and Microsatellite Haplotipes Point to a Single, Recent Entry of Native American Y-Chromosomes into the Americas // Moi. Biol. Evol. - 2004. - V. 21. - P. 164-175.

21. Zerjal T., Dashnyman B., Pandya A et al. Genetic relationship of Asians and Northern European, revealed by Y-chromocomal DNA analysis // Am. J. Hum. Genet. - 1997. - V. 60. - P. 1174-1183.

22. 1. Stepanov V.A., Kharkov B.H., Bubbles B.H. Evolution and phylogeny of the lines of the human Y-chromosome // Vestnik VOGiS. - 2006. - T. 10, No. 1. - S. 57-73.

Claims (3)

1. A method for determining haplogroups of a human Y-chromosome, comprising carrying out a multiplex polymerase chain reaction using a test system, and analyzing the resulting products on a DNA analyzer, characterized in that the method is carried out in two stages using a test system containing two sets : at the same time, at the first stage, genotyping is performed by multiplex PCR reaction using multiplexes of 20 specific oligonucleotide primers, presented in Table 2, for genotyping according to the main to the R, N, J, I, Q, C, E, D, G, O Y groups of the Y chromosome tree from the first set of the test system, the PCR amplification on the columns is purified, then a SNaPshot reaction is performed using multiplexes of 10- multiplexes by multiplex analysis These specific oligonucleotide primers are presented in Table 5 for genotyping according to the main haplogroups from the second set of the test system and the products are purified by TSAP enzyme, then in the second stage, if the mutation is detected in the first stage, a multiplex PCR reaction is performed using 62-fold multiplexesspecific oligonucleotide primers shown in Table 3, for typing by subhaplogroups from the first set of the test system and after purification of the amplification using columns, the SNaPshot reaction is carried out by multiplex analysis using multiplexes of the 28 specific oligonucleotide primers shown in Table 6 for typing according to subhaplogroups from the second set of the test system, and if no mutation is detected at the first stage, a multiplex PCR reaction is performed using multiplexes of 16 specific oligos nucleotide primers shown in Table 5, for typing on rare haplogroups A, C3, F, H, K, L, O3, T from the first set of test systems, after purification of the amplification using columns, the SNaPshot reaction is carried out by multiplex analysis using multiplexes For 8 specific oligonucleotide primers shown in Table 7, for typing according to rare haplogroups from the second set of test systems, the resulting products are purified with TSAP enzyme, and then they are analyzed on a DNA analyzer using the capillary gel electrophore method orez. 2. A test system for determining haplogroups of the human Y-chromosome, containing a set of reagents for PCR and a multiplex set of primers, characterized in that for the method according to claim 1, the test system additionally includes a second set for conducting SNaPshot reactions, the first set for PCR includes a mixture for the PCR reaction - 1.5 ml and three multiplex sets of specific oligonucleotide primers, each 1.5 ml: multiplexes of 20 specific oligonucleotide primers for genotyping according to the main Y-chromosome groups shown in Table 2, multiplexes of 62 specific oligonucleotide primers for genotyping by subgaplogroups, presented in Table 3, and multiplexes of 16 specific oligonucleotide primers for genotyping of rare haplogroups, presented in Table 4, a second SNaPshot reactions include three multiplex sets of specific oligonucleotide primers, each 1.5 ml each: multiplexes of 10 specific oligonucleotide primers for genotyping according to the main haplogroups Y-chromosomes shown in Table 5, multiplexes of 28 specific oligonucleotide primers for genotyping by subhaplogroups, presented in Table 6, and multiplexes of 8 specific oligonucleotide primers for genotyping of rare haplogroups, presented in Table 7. 3. Specific oligonucleotide primers for determining the haplogroups of the human Y chromosome for implementing the method according to claim 1, which are two sets of sequences, where in the first set of sequences shown in Tables 2,3 and 4, there is an annealing region of primers outside the mutation zones within species specificity with a melting point in the range of 55-64 ° C, GC composition of 40-60%; in the primer sequences there are no stable secondary structures - hairpins and dimers, and in the second set of sequences shown in Tables 5, 6 and 7, the annealing region of oligonucleotide primers is directly adjacent to the mutation zone within species specificity; in the primer sequences there are no stable secondary structures — studs and dimers; the calculated melting temperature for all primers is 50 ° C ± 2 ° C, GACT tails are attached to the 5 ′ ends of the primers according to the given oligonucleotide length.

Images