KR20110021095A - Snp markers for sex determination and/or human identification - Google Patents

Abstract

PURPOSE: A marker set for identifying sex and/or distinguishing individuals using SNP is provided to ensure usefulness of short amplicon and genotyping technique of high efficiency. CONSTITUTION: An SNP marker for identifying sex comprises three SNP markers for identifying sex detected in amelogenin gene(AMELX and AMELY) and three SNP markers detected in zinc finger protein gene(ZFX and ZFY). The SNP marker of amelogenin gene(AMELX and AMELY) is placed at base sequence #5139(C/T), #5196(C/G), and #5232(C/T). The SNP marker of zinc finger protein gene(ZFX and ZFY) is place at base sequence #60664(C/T), #60751(C/T), and #60766(A/G).

Description

 SNP markers for sex determination and / or human identification

The present invention relates to a marker for gender determination and / or individual identification using a single nucleotide polymorphism (SNP) marker having a single base mutation and a method for gender determination and / or individual identification using the same.

Gender and individual identification are important genetic testing methods widely used in criminal investigation science, and short tandem repeat (STR) markers, which have multiple alleles in various races, are mainly used. However, the use of STR markers has been limited due to problems such as high mutation rate, difficulty in obtaining a large amount of multiplexing information at once, and the need for amplification products of large size to determine alleles.

On the other hand, Single Nucleotide Polymorphism (SNP) refers to a genetic variation caused by a difference in a single base sequence in two different genomes existing between individuals, and represents the largest portion of all human genetic variants, that is, about 90% of all human genetic variants. It is a genetic mutation marker that accounts for the above.

SNP markers offer more compact and stable genetic maps than other genetically engineered markers, and are more suitable for new technologies, particularly high-throughput genotyping technology, including DNA chips. Have.

Therefore, the development of a new SNP marker to replace the STR marker currently used for identification of individuals with problems such as high mutation rate is expected to have many advantages and to be widely used in various fields.

An object of the present invention is to provide a sex discrimination SNP marker and / or individual identification SNP marker that can solve the problem of the existing STR marker.

Another object of the present invention is to provide a gender discrimination method and an individual identification method using a sex discrimination SNP marker and an individual identification SNP marker.

In order to achieve the above object, the present invention is a sex discriminated from three single nucleotide polymorphism (SNP) markers and zinc finger protein genes (ZFX & ZFY) for sex discrimination found in the Amelogenin gene (AMELX & AMELY) Provided are six gender discriminating SNP markers including three discriminating single nucleotide polymorphism (SNP) markers.

In addition, the present invention is 30 SNP markers for individual identification having a high degree of polymorphism in all ethnic groups, wherein the 30 SNP markers (GenBank Accession No at dbSNP) are rs7532151, rs4846468, rs6751657, rs10185531, rs7652776, rs6443222, rs17497475 rs1350191, rs2565007, rs4607417, rs1790006, rs2813838, rs2267708, rs1293288, rs7849782, rs7907658, rs550840, rs543840, rs734075, rs1151849, rs7328030, rs978511, rs716951, rs716 480 It provides a SNP marker for identifying an individual, characterized in that selected.

Hereinafter, the present invention will be described in more detail.

1. Gender Discrimination SNP Markers

Existing sex was determined using the Insertion / deletion (Indel) DNA marker present in the amelogenin gene (AMELX & AMELY), but in the present invention, the amelogenin gene (AMELX & AMELY) and zinc finger gene ( ZFX & ZFY) was used to determine the sex of humans using the difference in the single nucleotide sequence.

① Identifying SNP Markers for Gender Identification of Amellogenin Genes

First of all, two amelogenin genes (AMELX: present on the X chromosome and AMELY: Y chromosome) of each of the sex chromosomes X & Y and very similar gene sequences for sex discrimination using a single nucleotide sequence Sites with very similar sequences (see Fig. 3a) are first selected, followed by sequencing using DNA samples from 12 females and 12 males to identify three single base sequences (see Fig. 4a) that can fully differentiate sex. It was.

The three single nucleotide positions for which sex can be determined are shown in nucleotide sequence # 5139 (C / T) and nucleotide sequence # 5196 (C / G) in the AMELX gene sequence (GenBank Accession No: NC_000023) information registered in NCBI GenBank. , And located in nucleotide sequence # 5232 (C / T), and it was confirmed that the difference between the single nucleotide sequences accurately determined sex. At this time, the specific sequence information is shown in FIG.

② Identifying SNP markers for gender discrimination of zinc finger protein (ZFX & ZFY) genes

Sites with very similar nucleotide sequences of two zinc finger protein genes (ZFX: present on X chromosome & ZFY: Y chromosome), which are present on sex chromosomes X & Y, identically to the amelogenin gene (see FIG. 3B) Were first selected, and three single nucleotide sequences (see FIG. 4B) were identified to be completely distinguished by sequencing using DNA samples of 12 females and 12 males.

The three single nucleotide positions that can be determined by sex are shown in SEQ ID NO: # 60664 (C / T), SEQ ID NO: # 60751 (C / T), in the ZFX Gene Sequence (GenBank Accession No: NC_000023) information registered in NCBI GenBank. And it is located in the base sequence # 60766 (A / G) and it was confirmed that the difference between the single base sequence determines the sex. At this time, the specific sequence information is as shown in FIG.

In other words, in order to discover SNP markers for determining the sex of humans, multiple sequences of two genes on the X chromosome and the Y chromosome on the amelogenin gene (AMELX and AMELY) and the zinc finger protein genes (ZFX and ZFY), respectively Select regions with high homology. PCR primers can be used to amplify 150-250 bp of target region containing one nucleotide difference between genes on X and Y chromosomes (prime sequence is the base sequence of the underlined region in FIGS. 3A and 3B).

As an example, three single nucleotide sequence sites (AMEL-1, -2, -3) and zinc finger protein in the amelogenin gene were analyzed by sequencing of 24 individuals, including 12 males and 12 females. Three single nucleotide sequence sites (ZF-1, -2, -3) in the gene were able to fully distinguish between male and female (see Figures 4a and 4b).

In addition, sex may be determined by genotyping using one or two or more SNP marker sets among six SNP markers according to the present invention. In one embodiment, the selected AMEL-1 (C / T) site, AMEL-2 (C / G) site, and ZF-1 (C / T) from a plurality of subject DNA samples (total 960 DNA samples) The region was genotyped to exactly match the expected gender and genotype (see Figure 5).

 2. SNP Marker for Individual Identification

Conventional object identification methods are used STR markers, but in the present invention has been developed a method for identifying individuals using SNP markers for this purpose, the SNP markers for individual identification were identified using the following method.

First, a very high degree of polymorphism in Koreans was obtained using genotype information produced by 2,000 Koreans using Affymetrix's product (Genome-Wide Human SNP array 5.0), which can genotype 500,000 SNPs (500K) in total. A large number of SNP markers showing allele frequency> 0.45) were selected.

The selected SNP markers were finally selected from 56 SNP markers showing high polymorphism (minor allele frequency> 0.45) in all ethnic groups (Western, African & Oriental). Finally, 30 were selected from 56 selected SNP markers (Table 1), and the actual individual identification ability was tested using 960 Korean DNA samples.

As shown in Table 1, information of 30 SNP markers for identification of individuals (GenBank SNP ID: dbSNP) is as follows: rs7532151, rs4846468, rs6751657, rs10185531, rs7652776, rs6443222, rs17497475, rs1350191, rs2565007, rs4607417, rs138900, rs2267708, rs1293288, rs7849782, rs7907658, rs550840, rs543840, rs734075, rs1151849, rs7328030, rs978511, rs7164801, rs955665, rs4791495, rs4647887, rs1785745, rs7230112, rs818896.r

In addition, the present invention provides a sex discrimination and individual identification method capable of simultaneously identifying a sex and an individual by using a combination of the six sex discrimination SNP markers and the individual identification SNP markers.

The gender discrimination uses one or more gender discrimination markers, and the individual identification uses an individual identification SNP marker, wherein up to 100 individuals using 10 SNP markers and 15 SNP markers identify up to 1,000 individuals. Furthermore, when 20 SNP markers were used, it was possible to fully identify all 10,000 individuals (which were verified in 8,842 samples in the actual analysis).

Therefore, each time the number of individual identification samples increases 10 times, the number of individual identification SNP markers is increased by 5, and sex discrimination and individual identification can be determined using a DNA marker set for identification of individuals in various numbers of combinations.

In addition, if the number of SNP markers is increased and used up to 40, the Korean population has the same individual identification capability as the standard identification marker set using 16 STR markers.

On the other hand, the 30 selected SNP markers have a distance of at least 50 Mb (about 50 cM) apart from each other, and thus all have the same discrimination ability regardless of any combination.

In addition, in the present invention, genotype information related to gender discrimination, individual identification, and various other appearances (eg, skin color, hair color, and eye color, etc.) may be additionally used in genetic testing for at least one to a maximum of 386 SNP markers. At the same time, the use of Illumina's BeadExpress system, which can be genotyped, provides a way to add SNP markers related to various phenotypes in the future.

Gender determination and individual identification using the SNP marker according to the present invention, unlike the STR marker, has the advantages of low mutation rate, usefulness of amplicon size shorter than 100bp useful for digested DNA samples, and high efficiency genotyping technology. And very useful for identifying individuals.

The present invention is explained in more detail through the following examples. However, these examples are only for the purpose of embodying the subject matter of the present invention and the present invention is not limited by the examples.

All DNA samples used in the examples below were isolated from blood or Epstein Barr virus (EBV) -transformed B-cells according to standard DNA extraction methods. The following experiments were performed with the approval of the Asan Medical Center Institutional Review Board.

Example 1 Screening and Validation of SNP Markers for Gender Determination

1. Screening of SNP Markers for Gender Discrimination

In order to identify SNP markers for gender discrimination, DNA of 12 CEPH household samples (sample unique number: GM06994, GM07022, GM07347, GM07357, GM12962, GM12960, GM07000, GM07056, GM07346, GM07345, GM12863, GM12861) [Coriell Institute for Purchased from Medical Research (http://www.coriell.org/) and DNA samples of 12 Korean individuals (provided for use by the Genetic Resources Bank Team, Genome Center, National Institute of Health Research, Center for Disease Control and Prevention).

For sequencing of the amelogenin genes (AMELX and AMELY) and zinc pinker protein genes (ZFX and ZFY), genomic sequences were obtained from the GenBank (http: //www.ncbi/nlm.nih.gov/) database and PCR primers Was selected using Primer3 software (http://frodo.wi.mit.edu/) to amplify 150-250 bp of high homology target region containing one sequence difference between genes on X and Y chromosomes ( 3a and 3b). At this time, the PCR primer sequence is an underlined part.

PCR amplification reactions consisted of 20 ng genomic DNA, 0.15 μM each primer, 100 μM dNTP each, 20 units containing reaction buffer containing 1 unit AmpliTaq Gold ^TM (Applied Biosystems, Foster City, CA, USA) and 1.5 mM MgCl ₂ . It was performed in μl of solution. PCR was performed for 5 minutes at 94 ° C., followed by a total of 35 cycles of 45 seconds at 94 ° C., 30 seconds at 55 ° C., 1 minute at 72 ° C., and finally an extension reaction at 72 ° C. for 10 minutes. PCR products (1 μl) were used directly as templates for sequencing and sequenced according to manufacturer's instructions using a BigDye® terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) on an ABI3730 DNA sequencer. . DNA polymorphism was confirmed using the PolyPhred program (http://droog.gs.washington.edu/PolyPhred.html).

As a result, sequence comparisons of 24 individuals, including 12 males and 12 females, resulted in the amelogenin gene (AMEL-1, -2, -3) and zinc finger protein genes (ZF-1, -2,-). In 3), the male and female were completely distinguished by three (6 total) single nucleotide sequences each (see FIGS. 4A and 4B).

2. Evaluation of Effectiveness of SNP Marker for Gender Discrimination

In order to test the validity of the previously selected sex discriminating SNP markers, three sites (AMEL-1, AMEL-2 & ZF-1) were selected from a total of six sex determination single base sites. In this assay, genotyping of each SNP marker was performed according to the manufacturer's instructions using Illumina VeraCode GoldenGate analysis on a Korean DNA sample obtained with gender information (n = 960). Genotyping clusters and calling were performed using BreadStudio software. As a result, genotyping of the three markers, including AMEL-1, AMEL-2, ZF-1, was in perfect agreement with the expected gender genotype (see FIG. 5).

Example 2 Screening and Effectiveness Evaluation of SNP Markers for Individual Identification

1. Selection of SNP Markers for Individual Identification

To screen for SNP markers for identification, an Affymetrix 500K was first composed of 2,000 Korean SNP genotyping results using affymetrix's 500K SNP chip and allele frequency of four HapMap race samples (Japanese, Han Chinese, African, and European). SNP data ( Nat. Genet. 41, 527-534, 2009) was used.

Candidate SNP markers were selected based on the following SNP selection criteria. That is, (i) minor allele frequency (MAF)> 0.45 in all race groups, (ii) heterozygosity> 0.45, (iii) Hardy-Weinberg equilibrium (HWE) P -Value> 0.1, (iv) genotype call rate = 100%, and (v) physical distance between SNP markers> 50 Mb. Thirty SNPs were selected for efficacy evaluation based on the SNP selection criteria (Table 1).

2. Evaluation of the Effectiveness of SNP Markers for Individual Identification

Two different methods were used to validate the 30 SNP markers previously selected. In the first assay, 30 individual SNP markers were genotyped using DNA samples of 960 Koreans (n = 960). In another method, 8,842 Korean Affymetrix Genome-wide Human SNP array 5.0 data with 30 selected SNP markers were distributed and 30 individual SNP markers were used in a total of 8,842 Korean samples.

First, genotyping of SNP markers was performed according to the manufacturer's instructions using Illumina's VeraCode GoldenGate genotyping method on a set of 960 Korean DNA samples. Genotyping clustering and calling was performed using BreadStudio software.

Of the 30 SNP markers selected, 29 SNP markers were successfully genotyped and the other marker (rs6443222) was not genotyped. The allele and genotype frequencies of each SNP marker were found to be nearly identical to Table 1. As expected, all selected SNP markers showed even allele frequency. The total combined power of identity of the 29 SNPs was 4.83 × ^{10 −13} , which is consistent with the combined power of discrimination 0.999999999999517.

In the genotyping results of 960 people, only one pair (two individuals) showed the same genotype even when only 15 SNPs were used, which means that up to 1,000 individuals can be identified using 15 SNP markers. do.

In addition, according to the analysis of individual identification ability through Affymetrix SNP array 5.0 genotype data analysis including 8,842 individuals, the binding average coincidence probability of the 30 selected SNPs was 1.78X10 ^-13 , which was consistent with the binding identification 0.999999999999822. . As shown in Table 2, each time the number of SNP markers used to identify an individual increases by 5, the individual identification ability is increased by 10 times. That is, using 10 or 15 SNP markers can identify 100 and 1,000 individuals, respectively. In addition, when 20 SNP markers were used, all 8,842 individuals were fully identified.

SNP Count Number of samples analyzed (n) Number of samples representing the same genotype ^* % Average match 10 100 0-0-2 0.67 1,000 54-38-39 4.37 8,842 2,769 31.32 15 100 0-0-0 0.00 1,000 0-0-2 0.07 8,842 20 0.23 20 100 0-0-0 0.00 1,000 0-0-0 0.00 8,842 0 0.00

^For 100 and 1,000 samples, analytical samples were randomly selected three times from all samples (n = 8,842).

Figure 1 shows the position of the partial base sequence and sex discrimination sequence of the genome sequence NC_000023 of the AMELX gene,

Figure 2 shows the position of the partial base sequence and sex discrimination sequence of the genome sequence NC_000023 of the ZFX gene,

3A and 3B show the genomic DNA sequences of the amelogenin gene and the zinc finger protein gene, respectively,

Figures 4a and 4b shows the results of SNP identification in each of the amelogenin gene and zinc finger protein gene for sex determination,

Figure 5 shows the genotyping results of AMEL-2 markers using GoldenGate Assay in 960 individuals (purple dots represent males, blue dots represent females).

Claims (6)

Gender discrimination consisting of a total of six sexes, including three sex discrimination SNP markers found in the Amelogenin gene (AMELX & AMELY) and three sex discrimination SNP markers discovered in the zinc finger protein genes (ZFX & ZFY). SNP markers. The method according to claim 1, wherein the sex determination SNP markers of the amelogenin gene (AMELX & AMELY) is the base sequence # 5139 (C / T), in the AMELX gene sequence (GenBank Accession No: NC_000023) information registered in NCBI GenBank, SNP markers for gender determination, characterized by being located in nucleotide sequence # 5196 (C / G) and nucleotide sequence # 5232 (C / T). The method according to claim 1, wherein the SNP marker for gender determination of the zinc finger protein (ZFX & ZFY) gene is SEQ ID NO: # 60664 (C / T), in the ZFX gene sequence (GenBank Accession No: NC_000023) information registered in NCBI GenBank, SNP marker for gender determination, characterized in that located in nucleotide sequence # 60751 (C / T), and nucleotide sequence # 60766 (A / G). 30 SNP markers for highly discriminating individuals in all ethnic groups, wherein the 30 SNP markers (GenBank Accession No at dbSNP) are rs7532151, rs4846468, rs6751657, rs10185531, rs7652776, rs6443222, rs17497475, rs1350191, rs2565007, selected from rs4607417, rs1790006, rs2813838, rs2267708, rs1293288, rs7849782, rs7907658, rs550840, rs543840, rs734075, rs1151849, rs7328030, rs978511, rs7164801, rs955665, rs1794, rs178587 SNP markers for identifying individuals. Gender discrimination and individuals capable of identifying gender and individuals simultaneously, comprising combining six SNP markers for gender determination according to any one of claims 1 to 3 and 30 SNP markers for individual identification according to claim 4 Identification method. The method of claim 5, wherein the gender discrimination uses one or more gender discrimination markers, and the individual identification uses an individual identification SNP marker, but 100 samples use 10 SNP markers according to the number of samples to be identified. And 1000 samples using 15 SNP markers and 10,000 samples using 20 individual identification SNP markers, characterized in that sex identification and individual identification method.

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