A composite amplification kit for biogeographic ancestral DIPs and sex identification in degraded samples

Abstract

The present invention discloses a composite amplification cassette for biogeographic ancestry DIPs and sex identification for degraded specimen inference. The system is constructed based on a panel of 60 loci, including 56 DIPs loci on autosomes, 3 DIPs loci on Y chromosomes, and 1 enamel gene. Based on the panel combination, it is possible to distinguish between southern and northern East Asian populations on the basis of distinguishing intercontinental populations, and it can also be applied to forensic sex identification work. Because the amplicons of the 60 loci are all less than 200bp, the panel is suitable for the detection and analysis of highly degraded specimens.

Description

Technical Field

[0001] The invention belongs to the field of forensic medicine, and in particular relates to a composite amplification kit for biogeographic ancestry DIPs and sex identification inferred from degraded samples. Background Art

[0002] Forensic biogeographic ancestry inference, also known as ancestral inference, is to obtain the biogeographic ancestral origin of the sample to be tested or the proportion of the ancestral components of different biogeographical origins by detecting relevant indicators with population differences. In forensic applications, ancestral inference can provide direction for criminal investigations and provide evidence for judicial trials.

[0003] Detection of Ancestry Informative Markers (AIMs) is an effective means of forensic biogeographic ancestry inference. Deletion / Insertion Polymorphisms (DIPs) are widely distributed in the genome, have low mutation rates, and have racial and population differences. They play an important role in forensic ancestry inference and individual identification. The Human Genome Project (HGP), the International HapMap Project, and the Human Genome Diversity Project (HGDP) have described a large amount of DIPs site information, providing sufficient data for screening target AIM-DIPs for ancestry inference.

[0004] The existing AIM-DIPs system is mainly used to distinguish between African, East Asian, South Asian and European groups, and there are few studies on the distinction between groups within East Asia. China is located in East Asia, with a large north-south span and many ethnic groups. Effective methods to distinguish between southern and northern groups in East Asia will provide more accurate clues for case investigation. The existing SNP system is difficult to promote in grassroots forensic laboratories due to its long detection time and high cost. The DIP system constructed this time overcomes these defects and shortcomings and is more suitable for use in forensic laboratories in my country. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a DIPs composite amplification system for forensic biogeographic ancestry inference of degraded specimens. The present invention can be effectively used for: (a) distinguishing five major intercontinental groups; (b) distinguishing southern and northern groups in East Asia; (c) forensic gender identification through a set of molecular genetic marker compositions (a total of 60 site information and primers, including 56 DIPs sites on autosomes, 3 DIPs sites on the Y chromosome, and 1 enamel gene related to sex determination). The size of all amplicon fragments based on this method is within 77-199bp, and the detection system is suitable for specimens of various tissue types, especially solving the technical constraints of degraded specimen typing, and has a high application value in forensic ancestry inference, and can also be applied to forensic gender identification.

[0006] The first aspect of the present invention provides a gene panel combination, which includes 56 autosomal DIPs sites, 3 Y chromosome DIP sites and 1 enamel gene.

[0007] DIPs sites have the advantages of fast and convenient STR locus detection and low mutation rate and high stability of SNP sites, and are very suitable for application in the field of forensic genetics. Therefore, it is of great significance to combine AIM-DIPs to develop a DIPs ancestral inference system suitable for Chinese forensic grassroots DNA laboratories that can be applied to various types of specimens, especially degraded specimens.

[0008] In the present invention, the inventors used 3 DIPs sites on the Y chromosome and the Amelogenin site to determine the gender of samples of unknown origin; used 56 DIPs sites on the autosomes to infer the ancestry of intercontinental groups, and on this basis, effectively distinguished the southern and northern groups in East Asia, making it more suitable for use in my country's forensic DNA laboratories, providing more accurate clues for case investigation.

[0009] In the present invention, the three DIPs sites or enamel genes on the Y chromosome can more accurately identify gender.

[0010] According to the first aspect of the present invention, in some embodiments of the present invention, the 56 autosomal DIPs loci are rs34477782, rs4647655, rs35323999, rs66715534, rs2067350, rs141022607, rs3831885, rs33948716, rs34891898, rs3988320, rs3032990, rs4147539, rs10555216, rs3029066, rs3045215, rs10560368, rs 56038358, rs76533131, rs3028822, rs11273905, rs146391383, rs16432, rs3402443 8. rs5891435, rs10580743, rs10538061, rs35434967, rs5860664, rs34618829, rs343 83321, rs66471060, rs67853165, rs57406754, rs68108181, rs35171885, rs6718598 6. rs3840222, rs148177611, rs34530228, rs71997819, rs10534050, rs10569275, rs rs10533245, rs61044006, rs3842715, rs5868949, rs3835409, rs2307840, rs138324, rs5840834, rs35794676, rs3044252, rs5896844, rs71960622, rs3840794, and rs3033760.

[0011] In some embodiments of the present invention, the information of the 56 autosomal DIPs sites is shown in Table 1 of the specification.

[0012] In some embodiments of the present invention, the screening criteria for the autosomal DIPs site are: (1) in dbSNP, the lowest allele frequency is ≥ 0.1; (2) it is a di-allelic polymorphism of ins, del or delins; (3) the insertion or deletion sequence length is within 20 bp; (4) the candidate sites must be in linkage equilibrium, the physical distance between sites on the same chromosome must exceed 5 Mb, or they are located on different chromosomes; (5) the difference in allele frequency between populations is greater than 0.2; (6) it conforms to the Hardy-Weinberg equilibrium law.

[0013] In some embodiments of the present invention, the three Y chromosome DIPs sites are rs771783753, rs759551978 and rs2032678.

[0014] In some embodiments of the present invention, the information of the three Y chromosome DIPs sites is shown in Table 1 of the specification.

[0015] In some embodiments of the present invention, the gene panel combination is used for forensic biogeographic ancestry inference, forensic sex identification, and distinguishing five major continental populations and southern and northern East Asian populations.

[0016] In the present invention, the forensic ancestry inference system 60-panel (i.e., the gene locus panel combination described in the first aspect of the present invention) constructed based on the polymerase chain reaction (PCR) combined with capillary electrophoresis (CE) platform has good performance, with amplicon fragments within 200bp, high sensitivity and strong specificity, and is suitable for the analysis of forensic degraded samples.

[0017] The second aspect of the present invention provides a detection kit, wherein the detection kit contains the gene locus panel combination described in the first aspect of the present invention.

[0018] In some embodiments of the present invention, the product further comprises other adjuvants.

[0019] In some embodiments of the present invention, the auxiliary agents include but are not limited to PCR amplification reagents, such as dNTPs, buffers, enzymes, fluorescent markers, positive controls, negative controls and the like.

[0020] In some embodiments of the present invention, the positive controls include, but are not limited to, gender control samples, standard samples of five continental populations, standard samples of southern East Asian populations, and standard samples of northern East Asian populations.

[0021] The third aspect of the present invention provides a product for detecting loci in the gene locus panel combination described in the first aspect of the present invention, for use in any one of the following (1) to (3);

[0022] (1) Forensic biogeographic ancestry inference;

[0023] (2) Distinguishing between five continental groups and southern and northern East Asian groups;

[0024] (3) Forensic gender identification

[0025] In some embodiments of the present invention, the product includes specific primers, detection reagents, and detection kits.

[0026] In some embodiments of the present invention, the sequence of the specific primer is shown in SEQ ID NO:17~SEQ ID NO:137.

[0027] In some embodiments of the present invention, the design criteria of the specific primers are: it is necessary to confirm that there is no polynucleotide, no other insertion / deletion sequence, no genetic variation such as SNP in the site flanking sequence. It is also required that the length of the amplified fragment corresponding to the designed primer is less than 200 bp. The specificity of the primer sequence, the absence of special secondary structure of the primer, the stability of the primer binding to the target sequence and the primer amplification efficiency are ensured.

[0028] In some embodiments of the present invention, the product further comprises other adjuvants.

[0029] In some embodiments of the present invention, the auxiliary agents include but are not limited to PCR amplification reagents, such as dNTPs, buffers, enzymes, fluorescent markers and other substances.

[0030] In some embodiments of the present invention, the product analyzes and determines the data results of the DIPs site based on PCR composite amplification and CE platform.

[0031] In some embodiments of the present invention, the PCR amplification system is as shown in Table 3.

[0032] In some embodiments of the present invention, the PCR reaction program is: pre-denaturation at 95°C for 5 min; 94°C, 10 s, 63°C, 90 s, 2 cycles; 94°C, 10 s, 60°C, 90 s, 25 cycles; final extension at 60°C for 15 min.

[0033] In some embodiments of the present invention, the PCR amplification products are detected by fluorescence on a capillary electrophoresis genetic analyzer. After the capillary electrophoresis is completed, GeneMapper ® ID-X The software processes the detected data and obtains typing maps and data of 60 loci.

[0034] The fourth aspect of the present invention provides a method for distinguishing five major intercontinental groups and southern and northern East Asian groups, comprising the following steps: using primers shown in SEQ ID NO: 17 to SEQ ID NO: 137 to amplify the subject's sample, obtain the typing of three Y chromosome DIPs sites and enamel genes, and determine the subject's gender; obtain the genotyping of 56 autosomal loci, and use bioinformatics methods to determine the subject's intercontinental origin and whether it belongs to the southern or northern East Asian population.

[0035] In some embodiments of the present invention, the criteria for determining the subject's gender, continental origin, and whether the subject belongs to the southern or northern East Asian population are as follows: judging the gender through the typing results of the three DIPs sites on the Y chromosome and the enamel gene of the subject's sample; judging the subject's continental origin through bioinformatics analysis of the data of the 56 DIPs sites on the autosomes of the subject's sample and the data of the five major continental populations; judging the subject's ancestral origin through bioinformatics analysis of the typing data of the 56 DIPs sites on the autosomes of the subject's sample confirmed to be from East Asia and the data of the southern and northern East Asian populations.

[0036] In the present invention, based on the provided system, primers are designed for 56 DIPs sites on autosomes, 3 DIPs sites on the Y chromosome, and the tooth enamel gene Amelogenin, and six-color fluorescent labeling composite amplification is performed to effectively realize forensic biological geographic ancestry inference and gender identification. The amplicon fragment lengths of the 60 sites are all within 200bp, which can be used to type various types of forensic samples, especially degraded samples, and provide strong technical support for forensic ancestry inference and gender identification.

[0037] In some embodiments of the present invention, the five major continental groups are Africa, America, Europe, East Asia and South Asia.

[0038] In some embodiments of the present invention, the specific steps are:

[0039] The genomic DNA of the subjects was extracted and tested using the above-mentioned system (60-panel) to obtain the genotype data of 56 DIPs loci on the subjects' autosomes, 3 DIPs loci on the Y chromosome, and the Amelogenin locus;

[0040] The genotype data of the subjects at 56 DIPs loci on the autosomes were analyzed by bioinformatics with the data of five continental populations to determine the continental origin of the subjects;

[0041] The genotype data of the subjects from East Asia at the 56 DIPs loci on the autosomes were analyzed by bioinformatics with the data of the southern and northern populations of East Asia to determine whether the subjects belonged to the southern or northern part of East Asia;

[0042] The genotypes of the three DIPs sites and the Amelogenin site on the subject's Y chromosome were analyzed to determine the subject's gender.

[0043] In some embodiments of the present invention, the determination method can be performed by referring to conventional methods in the art, or the results can be obtained by using software analysis.

[0044] The beneficial effects of the present invention are:

[0045] The allele frequencies of the 56 autosomal DIPs sites selected by the present invention vary greatly between intercontinental and northern and southern Asian populations, and are suitable for the identification of intercontinental populations, and can distinguish between southern and northern East Asian populations on the basis of distinguishing intercontinental populations, and can also accurately identify gender at the same time. The present invention adopts a six-color fluorescent labeling technology to achieve stable, sensitive, specific, and efficient composite amplification of 56 DIPs sites on autosomes, 3 DIPs sites on Y chromosomes, and gender identification gene Amelogenin, thereby improving the identification efficiency of the system, so that the molecular genetic marker site combination can be effectively applied to forensic ancestral inference and gender identification work. The present invention simultaneously amplifies the autosomal and Y chromosome DIPs sites, and uses three Y-DIPs sites on the Y chromosome that are not located near the Amel-Y region and the common microdeletion region, so that gender can be more accurately identified. Through the allele typing data of the 56 DIPs loci in the present invention, the accuracy of ancestral inference can be evaluated based on cross-validation, and the amplicon lengths of the 60 loci are all within 200bp, which can increase the site detection rate of degraded samples and is suitable for the detection and analysis of highly degraded samples. The detection method in the present invention is simple and can use a commonly used capillary electrophoresis genetic analyzer, which is easy to promote and apply in grassroots forensic DNA laboratories. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The typing result of 1 ng DNA standard 9948 was detected using the present invention.

[0047] Figure 2 A~2F are PCA plots based on 56 DIPs loci on autosomes. AFR represents the African population, EUR represents the European population, EAS represents the East Asian population, SAS represents the South Asian population, AMR represents the American population, JPT represents the Japanese population, CDX represents the Xishuangbanna Dai population in China, KHV represents the Kinh population in Vietnam, and CHH represents the Han population in China.

[0048] Figure 3 This is a STRUCTURE analysis diagram based on 56 DIPs loci on autosomes, Figure 3 A is the STRUCTURE analysis chart of 26 groups around the world. Figure 3 B is the STRUCTURE analysis diagram of five East Asian groups, among which CHB represents the Han Chinese group in Beijing, China, and CHS represents the Han Chinese group in southern China.

[0049] Figure 4These are the evaluation results of the accuracy of ancestry inference based on cross-validation, where A is the evaluation result of the five continents; B is the evaluation result of subdividing the East Asian group into North East Asia and South East Asia.

[0050] Figure 5 The existing system (Y-STR Panel) is used to classify degraded samples

[0051] Figure 6 This is the situation of the 60-panel classification of the degradation sample in the present invention. DETAILED DESCRIPTION

[0052] In order to make the invention purpose, technical solution and technical effect of the present invention clearer, the present invention is further described in detail below in conjunction with specific implementation methods. It should be understood that the specific implementation methods described in this specification are only for explaining the present invention, not for limiting the present invention.

[0053] Unless otherwise specified, the experimental materials and reagents used are consumables and reagents that can be obtained from conventional commercial channels.

[0054] Construction of DIP multiplex amplification system

[0055] Based on the SNP database (Build 154) and previous literature reports, the autosomal DIP loci required in the embodiments of the present invention were screened according to the following criteria:

[0056] (1) In dbSNP, the lowest allele frequency of the selected DIP locus is ≥ 0.1;

[0057] (2) diallelic polymorphisms of ins, del, or delins;

[0058] (3) The insertion / deletion sequence length is within 20 bp;

[0059] (4) The candidate loci must be in linkage equilibrium, with the physical distance between loci on the same chromosome greater than 5 Mb, or located on different chromosomes;

[0060] (5) The difference in allele frequency between populations is greater than 0.2;

[0061] (6) It complies with the Hardy-Weinberg equilibrium law.

[0062] A total of 56 autosomal loci were screened, including rs34477782, rs4647655, rs35323999, rs66715534, rs2067350, rs141022607, rs3831885, rs33948716, rs34891898, rs3988320, rs3032990, rs4147539, rs10555216, rs3029066, rs3045215, rs10560368, rs 56038358, rs76533131, rs3028822, rs11273905, rs146391383, rs16432, rs3402443 8. rs5891435, rs10580743, rs10538061, rs35434967, rs5860664, rs34618829, rs343 83321, rs66471060, rs67853165, rs57406754, rs68108181, rs35171885, rs6718598 6. rs3840222, rs148177611, rs34530228, rs71997819, rs10534050, rs10569275, rs rs10533245, rs61044006, rs3842715, rs5868949, rs3835409, rs2307840, rs138324, rs5840834, rs35794676, rs3044252, rs5896844, rs71960622, rs3840794, and rs3033760.

[0063] At the same time, the inventors also introduced four sites for sex identification, including three DIPs sites on the Y chromosome and one enamel gene Amelogenin related to sex determination. Among them, the three DIPs sites on the Y chromosome are rs771783753, rs759551978 and rs2032678.

[0064] The detailed information of the above 60 genomes is shown in Table 1.

[0065] Table 1 Relevant information of 60 loci

[0066]

[0067] In the table, Bn, Gn, Yn, Rn, and Pn are different sites labeled with blue, green, yellow, red, and orange fluorescence, respectively; Chrom is the abbreviation of Chromosome.

[0068] Primers are designed for the selected sites. It is necessary to confirm that there are no polynucleotides, no other insertion and deletion sequences, and no genetic variations such as SNPs in the flanking sequences of the sites. The amplicon length corresponding to the primers is required to be less than 200bp. At the same time, the species specificity of the primer sequence, the formation of primer dimer structure, the stability of the primer binding to the target sequence, and the primer amplification efficiency are guaranteed.

[0069] The primer information corresponding to the above genome is shown in Table 2.

[0070] Table 2 Primer information of 60 loci

[0071]

[0072] The primer concentrations in Table 2 refer to the sum of the concentrations of the forward primer and the reverse primer in each set of primers.

[0073] Based on the above primers, a PCR composite amplification system is constructed to test the test samples. The specific steps are as follows: prepare the human sample to be tested, which can be genomic DNA extracted from human body fluids / tissues (such as blood stains, blood, saliva, oral swabs, hair roots with hair follicles, semen, muscle tissue, exfoliated cells, etc.). The extraction method can be the Chelex-100 method, phenol chloroform method, magnetic bead method, etc. for extraction and quantification; it can also be direct amplification without extraction of a variety of test materials (such as blood, blood filter paper, blood gauze, FTA card, hair roots with hair follicles, saliva card, exfoliated cells, etc.). The amplification system is shown in Table 3.

[0074] Table 3 Multiplex amplification system

[0075]

[0076] The dosage of human biological samples can refer to the following dosage standards: for DNA, the dosage is 1µL; for direct amplification samples (such as tissues), the dosage is 1.0-1.2mm 2 ; For other samples, judge based on normal circumstances.

[0077] The specific components of Master mix include Taq enzyme, Tris-HCl buffer, KCl, MgCl 2 And BSA, etc.

[0078] The amplification reaction program was as follows: pre-denaturation at 95°C for 5 min; 2 cycles of 94°C, 10 s, 63°C, 90 s; 25 cycles of 94°C, 10 s, 60°C, 90 s; and final extension at 60°C for 15 min.

[0079] The amplified products were detected by fluorescence on a capillary electrophoresis genetic analyzer. After the capillary electrophoresis, GeneMapper ® ID-X The software processes the detected data and obtains typing maps and data of 60 loci.

[0080] Figure 1 This is the genotyping map obtained by testing the sample using 1ng DNA standard 9948. It can be seen that the above method can be used for effective typing of DNA samples.

[0081] Application of the method in the above embodiment in forensic family origin inference

[0082] In this embodiment, the inventors selected 2504 human data from 26 populations in the public database 1000 Genomes as validation samples for analysis to verify the effectiveness of the above method.

[0083] The specific steps are:

[0084] The principal component analysis (PCA) was performed using the online software STRAF, the population genetic structure analysis was performed using the STRUCTURE v2.3.4 software, the stacking plots were drawn using Distruct 1.1, and the accuracy of ancestral inference was evaluated using cross-validation.

[0085] (1) Principal component analysis:

[0086] The principal component analysis results are as follows Figure 2 shown.

[0087] Depend on Figure 2 It can be seen that when PCA analysis was performed on individuals from Africa, Europe, and Asia using the 56 DIPs loci on the autosomes, individuals from the same continental origin were clustered together, and the distinction between different continents was high ( Figure 2 A); When samples from Africa, Europe, Asia, America, and South Asia were analyzed simultaneously using PCA, the Americas, South Asia, and Europe were not sufficiently distinguished ( Figure 2 B); when the samples from the above three continents were analyzed separately, the samples from America, South Asia and Europe showed better clustering effects ( Figure 2 C). This result shows that the system is more effective in distinguishing Africa, Europe and East Asia than South Asia, Europe and America, suggesting that when conducting PCA analysis, choosing a reasonable reference group will make the interpretation of the results more accurate. The PCA results of the East Asian internal group show that the Japanese (JPT) in Northeast Asia and the Xishuangbanna Dai (CDX) in China near Southeast Asia can be distinguished ( Figure 2D); the Han ethnic group, due to its north-south gradient variation, is between JPT and CDX, and has varying degrees of overlap with both ( Figure 2 E); the Vietnamese Kinsman (KHV) in Southeast Asia overlaps more with CDX ( Figure 2 F). This result shows that this system has a good ability to distinguish representative groups in southern East Asia and northern East Asia, but cannot distinguish groups in southern or northern East Asia.

[0088] (2) STRUCTURE analysis:

[0089] STRUCTURE analysis results are as follows Figure 3 shown.

[0090] STRUCTURE analysis was performed on the above samples using data from 56 DIPs loci on autosomes. Figure 3 As shown in A, when K =5, Africa, Europe, East Asia, South Asia and America all showed unique ancestral components, but the CLM and PUR populations in America showed more ancestral components consistent with Europe; Figure 3 As shown in B, when K =5, the main ancestral component of the northern East Asian group JPT is displayed in blue, and the main ancestral component of the southern East Asian group CDX is displayed in brown. The ancestral components represented by these two colors show a clear gradient change from north to south in the five subgroups.

[0091] (3) Cross-validation to evaluate the accuracy of ancestry inference:

[0092] The cross-validation ancestry inference accuracy evaluation results are as follows Figure 4 shown.

[0093] The accuracy of ancestry inference was evaluated using the typing data of 56 DIPs loci on autosomes of 2504 samples. When inferring intercontinental groups in Africa, Europe, East Asia, South Asia, and the Americas, the external validation accuracy of East Asian ancestry inference was 100% ( Figure 4 A), when East Asia was further divided into North East Asia and South East Asia, the external validation accuracy was 82.04% and 83.89%, respectively ( Figure 4 B).

[0094] Application of the method in the above embodiment in forensic degradation sample inspection

[0095] Five degraded samples confirmed by commercial STR kits were taken, and the DNA of the five degraded samples was amplified using the method (complex amplification system) in the above embodiment. The obtained products were subjected to capillary electrophoresis and further typing.

[0096] The results are as follows Figure 5 , 6 shown.

[0097] It can be found that the typing results obtained by using the method in the above embodiment (DIPs composite amplification system) are compared with the test results of the commercial STR kit. It can be found that the site detection rate and typing reliability of the degraded sample using the method in the above embodiment are higher.

[0098] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A product for detecting a combination of gene panels, characterized in that: The gene panel combination is a combination of 56 autosomal DIPs loci, 3 Y chromosome DIPs loci and 1 tooth enamel gene Amelogenin; The 56 autosomal DIPs loci are rs34477782, rs4647655, rs35323999, rs66715534, rs2067350, rs141022607, rs3831885, rs33948716, rs34891898, rs3988320, rs3032990, rs4147539, rs1055521 6. rs3029066, rs3045215, rs10560368, rs56038358, rs76533131, rs3028822, rs11273905, rs146391383, rs16432, rs34024438, rs5891435, rs10580743, rs10538061, rs35434967, rs 5860664, rs34618829, rs34383321, rs66471060, rs67853165, rs57406754, rs68108181, rs 35171885, rs67185986, rs3840222, rs148177611, rs34530228, rs71997819, rs10534050, r s10569275, rs10533245, rs61044006, rs3842715, rs5868949, rs3835409, rs2307840, rs138324, rs5840834, rs35794676, rs3044252, rs5896844, rs71960622, rs3840794, and rs3033760; The three Y chromosome DIPs sites are rs771783753, rs759551978 and rs2032678.

2. The product according to claim 1, characterized in that The product is used for forensic biogeographic ancestry inference and differentiation of five major continental populations and southern and northern East Asian populations.

3. A detection kit, characterized in that: The detection kit contains the product according to any one of claims 1 to 2.

4. Use of the product according to any one of claims 1 to 2 in any one of the following (1) to (2); (1) Forensic biogeographic ancestry inference; (2) Distinguish between five major continental groups and southern and northern East Asian groups.

5. The use according to claim 4, characterized in that: The product includes a detection reagent.

6. The use according to claim 5, characterized in that: The product includes specific primers, and the sequences of the specific primers are shown in SEQ ID NO:17 to SEQ ID NO:

137.

7. A method for distinguishing five continental groups and southern and northern East Asian groups, comprising the following steps: The primers shown in SEQ ID NO:17 to SEQ ID NO:137 were used to amplify the subject samples, obtain the subject's typing data, and determine the subject's gender. The relevant population genetics parameters and forensic parameters were calculated and compared with the reference group to determine the subject's continental origin and whether he or she belonged to the southern or northern East Asian population.

8. The method according to claim 7, characterized in that The criteria for determining the subject's gender, continental origin, and whether they belong to the southern or northern East Asian population are: The gender of the subject is determined by the typing results of the three Y chromosome DIPs loci and the tooth enamel gene Amelogenin. The intercontinental origin of the subjects was determined by bioinformatics analysis of the typing data of 56 autosomal DIPs loci of the subject samples and the intercontinental population data; The biogeographical origin of the subjects was determined by confirming the genotyping of 56 autosomal DIPs loci in samples from East Asian subjects and conducting bioinformatics analysis with data from southern and northern East Asian populations; The three Y chromosome DIPs sites are rs771783753, rs759551978 and rs2032678; The 56 autosomal DIPs loci are rs34477782, rs4647655, rs35323999, rs66715534, rs2067350, rs141022607, rs3831885, rs33948716, rs34891898, rs3988320, rs3032990, rs4147539, rs1055521 6. rs3029066, rs3045215, rs10560368, rs56038358, rs76533131, rs3028822, rs11273905, rs146391383, rs16432, rs34024438, rs5891435, rs10580743, rs10538061, rs35434967, rs 5860664, rs34618829, rs34383321, rs66471060, rs67853165, rs57406754, rs68108181, rs 35171885, rs67185986, rs3840222, rs148177611, rs34530228, rs71997819, rs10534050, r s10569275, rs10533245, rs61044006, rs3842715, rs5868949, rs3835409, rs2307840, rs138324, rs5840834, rs35794676, rs3044252, rs5896844, rs71960622, rs3840794, and rs3033760.

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