CN108642190B

CN108642190B - Forensic medicine composite detection kit based on 14 autosomal SNP genetic markers

Info

Publication number: CN108642190B
Application number: CN201810644104.4A
Authority: CN
Inventors: 侯一平; 李介男; 叶懿; 宋凤; 罗海玻; 谢明坤; 郎敏; 王正; 钱小钦; 李英碧
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2018-06-21
Filing date: 2018-06-21
Publication date: 2021-02-09
Anticipated expiration: 2038-06-21
Also published as: CN108642190A

Abstract

The invention belongs to the technical field of forensic medicine, and particularly relates to a forensic medicine composite detection kit based on 14 autosomal SNP genetic markers. Aiming at the problem that the existing market lacks a forensic medicine detection kit suitable for female examination materials, the invention provides a forensic medicine composite detection kit based on 14 autosomal SNP genetic markers. The kit comprises: comprises a separately packaged composite amplification primer mixture, a multiple single-base extension reaction primer mixture and standard DNA. The kit applies composite amplification and single base extension technology, can obtain 14 autosomal chromosomes of biological detection materials at one time for genetic typing of the plateau adaptive SNP genetic marker, and can rapidly distinguish plateau crowds from low-altitude crowds.

Description

Forensic medicine composite detection kit based on 14 autosomal SNP genetic markers

Technical Field

The invention belongs to the technical field of forensic medicine, and particularly relates to a forensic medicine composite detection kit based on 14 autosomal SNP genetic markers.

Background

The inference of the source of the human biological material has very important significance in the actual medical examination case. By detecting SNP sites with obvious allele frequency difference among different populations, the geographic source of the human biological material can be deduced. The method has the advantages that the geographical source of the detected material can be accurately deduced, the investigation range can be reduced, the labor and material resources are reduced, the working efficiency is improved, and the case investigation is assisted.

The plateau population refers to the population with the altitude of the long-term living area above 2500 m. The number of the Chinese plateau population is close to 500 ten thousand, and the population is intensively distributed on the Qinghai-Tibet plateau and the surrounding areas. The high altitude environment has the characteristics of cold and low oxygen partial pressure, and the oxygen partial pressure of a plateau area with the altitude of 4000 meters is 60 percent of that of a low altitude area. Low oxygen partial pressure is likely to cause various high altitude diseases such as pulmonary edema, cerebral edema and erythrocytosis. The population living in high altitude area of generation will produce corresponding genetic change under the selective pressure, thereby adapt to the oxygen deficiency environment of plateau. Compared with the low-altitude population (the altitude of a long-term living area is below 2500 m), the plateau population in China has better aerobic exercise capacity, higher oxygen transportation efficiency, higher resting ventilation, higher blood oxygen saturation and hemoglobin level which does not increase obviously along with the increase of the altitude. The series of changes all have corresponding genetic basis. Research finds that the hypoxia inducible factor plays a key role in the process that the plateau population adapts to the plateau hypoxia environment. The whole genome association research finds SNP loci with significant difference in allele frequencies between a large number of high-altitude people and low-altitude people on coding genes of hypoxia inducible factors such as EPAS1 and EGLN1, wherein the SNP loci with the most significant difference in allele frequencies of the SNP loci of the EPAS1 gene are considered by scholars to be the most extreme examples of human adaptation to natural environments. The SNP loci with significant difference in allele frequencies between the plateau population and the low-altitude population on the EPAS1 gene can be used for deducing whether the biological test material of the human is from the plateau population or the low-altitude population.

Single Nucleotide Polymorphism (SNP) refers to DNA sequence Polymorphism caused by Single base mutation at a specific Nucleotide position in a genome, and includes insertion, deletion, transition, transversion and the like of a Single base. The SNP marker is considered as a third-generation novel molecular marker following SSR, ISSR and other markers, and has the characteristics of low mutation rate, high genetic stability, wide distribution, rich site information and the like. Research on the application of SNP markers in forensic personal identification, paternity testing, and test material source inference has become a focus of research in recent years.

Currently, SNP detection technologies include single-base extension reaction, massively parallel sequencing, Sanger sequencing and SNP chip technologies. Massively parallel sequencing can find more SNPs, but it is costly, has sampling errors, and its accuracy is affected by the depth and width of the sequence. And Sanger sequencing steps are complicated, the cost is high, the time consumption is long, and the dependence on the quality and the content of the DNA of a sample to be detected is strong, so that the Sanger sequencing method is difficult to widely apply. In the field of forensic medicine, single base extension reaction technology has been widely applied to typing of SNP genetic markers. Compared with a first generation sequencing method, the technology can compositely detect a plurality of SNP at one time, has the advantages of less required template DNA amount, small amplified fragment (less than 200bp) and the like, and is particularly suitable for detecting bones, hair samples, degraded test materials and trace test materials. Compared with large-scale parallel sequencing, the method has the advantages of low cost, simple operation and low false positive rate. The technology can be used for detection by means of a platform-capillary electrophoresis platform which is widely used in a forensic medicine genetic laboratory, and is easy to popularize. Therefore, forensic researchers mostly adopt a single base extension reaction technology to establish a forensic detection system capable of simultaneously typing multiple SNP sites.

At present, a forensic medicine composite detection kit based on 55Y chromosome SNP genetic markers and a forensic medicine composite detection kit based on 56Y chromosome SNP genetic markers are researched and developed, and the kits can correctly belong Chinese male individual samples to a high-resolution Y chromosome phylogenetic tree so as to infer the ethnic group source of unknown male test materials. Since these kits use Y chromosome SNP genetic markers, they cannot be applied to the case of female test materials, and meanwhile, these kits focus on the estimation of the family source of the test materials, and cannot determine the geographic source of the test materials, there is still a need to develop a detection kit capable of rapidly detecting the family source or geographic source of female test materials.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: the problem that a forensic medicine detection kit suitable for female inspection materials is lacked in the existing market is solved.

The technical scheme for solving the technical problems comprises the following steps: provides a forensic medicine composite detection kit based on 14 autosomal SNP genetic markers. The kit comprises the following components: separating the packaged composite amplification primer mixture, the multiple single-base extension reaction primer mixture and the standard DNA; the number of the composite amplification primer mixtures is 28; the primer mixture of the multiple single-base extension reaction has 14 pieces.

Wherein, in the forensic medicine composite detection kit based on 14 autosomal SNP genetic markers, the 14 autosomal SNP loci are shown in Table 1.

TABLE 114 autosomal SNP loci Table

In the forensic medicine composite detection kit based on 14 autosomal SNP genetic markers, the nucleotide sequences of the amplification primers in the composite amplification primer mixture are shown as SEQ ID No.1-SEQ ID No.28 in Table 2.

TABLE 2 composite amplification primer mixture sequences

In the forensic medicine composite detection kit based on 14 autosomal SNP genetic markers, the nucleotide sequences of the primers in the multiple single-base extension reaction primer mixture are respectively shown as SEQ ID No.29 to SEQ ID No.42 in Table 3:

TABLE 3 Single-base extension primer mixture sequences

In the above table, the "-" symbol is preceded by a tailing sequence.

The forensic medicine composite detection kit based on 14 autosomal SNP genetic markers further comprises standard DNA, wherein the standard DNA is 2800M standard DNA.

The invention also provides application of the forensic medicine composite detection kit based on the 14 autosomal SNP genetic markers in distinguishing the high-altitude population from the low-altitude population.

The plateau population refers to the population living at the altitude higher than 2500 m for a long time; the low-altitude crowd refers to the crowd living at the altitude of less than or equal to 2500 m for a long time.

The invention also provides a use method of the forensic medicine composite detection kit based on the 14 autosomal SNP genetic markers, which comprises the following steps:

a. extracting DNA of a sample to be detected as an amplification template;

b. b, performing two-system composite amplification on the DNA extracted in the step a by adopting a composite amplification primer mixture and a composite amplification reaction mixed solution;

c. purifying the composite amplification product in the step b, and performing multiplex single-base extension reaction of two systems by using the composite amplification product as a template and using a multiplex single-base extension primer mixture and a single-base extension reaction mixed solution;

d. purifying the product of step c with Shrimp Alkaline Phosphatase (Shrimp alkali Phosphatase SAP), performing capillary electrophoresis analysis, and obtaining genotyping result of the sample according to the electrophoresis result.

In the use method, the cycle parameters of the reaction of the multiplex amplification PCR in the step b are as follows: 15 minutes at 95 ℃; Touchdown-PCR (Touchdown-PCR) for 12 cycles at 94 ℃, 30 seconds, 66 ℃ to 60 ℃, 90 seconds, 72 ℃, 30 seconds; 94 ℃, 30 seconds, 60 ℃, 90 seconds, 72 ℃, 30 seconds, 17 cycles; then, the temperature is 72 ℃ for 10 minutes; storing at 4 ℃.

Wherein, in the using method, the cycle parameters of the single base extension reaction in the step c are as follows: 94 ℃, 10 seconds, 50 ℃, 5 seconds, 60 ℃, 30 seconds, 26 cycles and then storage at 4 ℃.

The invention has the beneficial effects that:

the kit comprises functional SNP genetic markers which are related to adaptation of plateau hypoxia environments of plateau swarms on 14 autosomal EPAS1 genes, wherein allele frequencies of the SNP markers are obviously different between plateau crowds and low-altitude crowds, and the kit can be used for deducing whether a human biological material of unknown origin belongs to the plateau crowds or the low-altitude crowds in a forensic actual detection case;

the kit applies the single-tube composite amplification and multiple single base extension technology, can obtain the genotyping of 14 autosomal SNP genetic markers of the biological test material at one time, and can simply and quickly deduce the source of the biological test material in forensic medicine; the kit comprises standard DNA2800M to ensure the accuracy and reliability of the typing results. The length of the composite amplification product of the kit is only 80bp at the shortest and not more than 220bp at the longest, and the kit has advantages for the detection of common degradation detection materials in the actual detection case of the forensic science; the kit takes the universal capillary electrophoresis of the forensic genetic laboratory as a detection platform, and has wide application and popularization values.

Drawings

FIG. 1 shows the result of electrophoresis typing based on the 14 autosomal plateau adaptive SNP genetic marker kit. b is the typing result of the standard DNA 2800M; a and c are the typing results of plateau samples. The abscissa value in the figure indicates the DNA chain length, the ordinate value indicates the fluorescence intensity, and the SNP genetic marker represented by each allele peak is indicated above the peak.

Detailed Description

The invention provides a forensic medicine composite detection kit based on 14 autosomal SNP genetic markers, which comprises the following components: separating the packaged composite amplification primer mixture, the multiple single-base extension reaction primer mixture and the standard DNA; the number of the composite amplification primer mixtures is 28; the primer mixture of the multiple single-base extension reaction has 14 pieces.

The kit is based on functional SNP genetic markers related to adaptation of plateau population to plateau anoxic environment on 14 autosomal EPAS1 genes obtained by screening, and a plateau adaptation SNP genetic marker composite detection system is constructed by a capillary electrophoresis detection platform and by utilizing composite PCR amplification and single base extension technologies.

The working principle of the kit is as follows: firstly, carrying out PCR amplification on a sample to be detected through a composite amplification primer mixture and a composite amplification reaction mixed solution to obtain a DNA fragment containing 14 autosomal plateau adaptive SNP markers. And then taking the amplification products, namely 14 DNA fragments, as templates, and carrying out multiple single-base extension reactions by using the single-base extension reaction mixed liquor and the corresponding single-base extension reaction primer mixed liquor to obtain single-base extension reaction products of the 14 autosomal plateau adaptive SNP markers of the sample to be detected. Mixing the single base extension product and formamide containing an internal standard according to a certain proportion, performing capillary electrophoresis, and determining the allele represented by each allele peak on a typing map by using the fragment length of the single base extension reaction product of each SNP site and the mutation type of the SNP site, thereby obtaining the typing result of the 14 autosomal plateaus to be detected, which are adaptive to the SNP marker.

In addition, 2800M standard DNA was used as a positive control to perform amplification and extension reactions simultaneously with the sample. And whether the detection result is reliable can be judged according to whether the typing result of the standard DNA is accurate or not.

In particular, the key point of the invention is that 14 autosomal plateau adaptive SNP marker loci are selected.

The high altitude environment has the characteristics of cold and low oxygen partial pressure, and the population living in the high altitude area in generation can generate corresponding genetic change under the selection pressure, so that the high altitude environment is adapted. The whole genome association research publishes a large number of Tibetan plateau adaptation related SNP sites which are distributed on hypoxia-inducible factor (HIF) encoding genes such as EPAS1, EGLN1, PPARA, HMOX2, HBG2 and PKLR, wherein the frequency of SNP alleles on EPAS1 and EGLN1 is most different in plateau population and low-altitude population.

However, not all sites on these genes can effectively distinguish high altitude from low altitude populations. According to the invention, the allele frequencies of EPAS1 and EGLN1 gene plateau adaptive SNP loci in Tibetan population are compared with 26 populations of thousand-people genome plan database, and the frequency difference of different plateau adaptive SNP loci in plateau population and low-altitude population is different. For example, the rs12097901 locus of the EGLN1 gene related to Tibetan plateau adaptation is reported in the literature, the frequency of the derived allele is 68% in the Tibetan plateau population, and is 42% -49% in the east Asia population of the thousand-people genome planning database, so the locus has little significance in the research of distinguishing the plateau population from the low-altitude population; in contrast, the rs150877473 site on the EPAS1 gene has the frequency of approximately 70% in the population of the high altitude Tibetan family, and the frequency of less than 5% in the population of the thousand human genome project database, and the site can be used for the research of distinguishing the high altitude population from the low altitude population.

The SNP locus selected in the kit has high derivative allele frequency in plateau population, and the derivative allele frequency in low-altitude population is extremely low. Therefore, through a large number of analyses and studies, a screening standard for autosomal plateau adaptive SNP markers incorporated into the kit of the present invention was established: locating in the intron region of the hypoxia inducible factor coding gene; the frequency difference of the derivative allelic base in the plateau population and the low-altitude population is more than 50 percent; thirdly, the flanking sequence of the SNP locus can be designed into a proper composite amplification primer and a multiple single base extension primer; and fourthly, obtaining stable typing results by using a single base extension technology. According to the standard, 14 autosomal plateau adaptive SNP loci are screened out finally. The 14 autosomal plateau adaptive SNP locus information related in the 14 autosomal plateau adaptive SNP genetic marker detection kit is shown in Table 4.

Table 414 autosomal plateau adaptive SNP loci

Serial number	SNP loci	Nucleotide localization	Polymorphism
					1	rs115321619	Chr2:46567916	G->A
2	rs13419896	Chr2:46556345	G->A
				3	rs73926265	Chr2:46569770	G->A
4	rs55981512	Chr2:46570342	G->A
				5	rs116611511	Chr2:46600030	A->G
6	rs189807021	Chr2:46583581	G->A
				7	rs372272284	Chr2:46584859	A->G
8	rs374487821	Chr2:46571435	G->C
				9	rs73926264	Chr2:46569017	A->G
10	rs150877473	Chr2:46588019	C->G
				11	rs73926263	Chr2:46568680	A->G
12	rs149306391	Chr2:46571017	C->G
				13	rs369097672	Chr2:46600358	A->G
14	rs142826801	Chr2:46588331	G->C

In the above table, the nucleotide positions are the positions of the SNP sites in the hg19 version.

The kit disclosed by the invention applies a composite PCR amplification technology in the detection of the SNP of the Y chromosome. The composite PCR amplification technology can amplify a plurality of target DNA fragments in one reaction system, has the advantages of convenience, rapidness, sample saving and low cost, and is suitable for the requirement of actual case detection of forensic medicine. The design of the composite PCR amplification primer is the key and difficult point of the technology, and the invention fully considers the following factors when designing the primer: the GC ratio of the primer is within the range of 30-70 percent; annealing temperature of all primers is approximate; ③ the length range of the primer is 18 to 30 bases; fourthly, the length range of the amplified product is between 80bp and 220bp, so as to be suitable for the detection of the degraded detection material; fifthly, no obvious mismatching exists among the primers, the primers and the template, and a hairpin structure and a dimer structure are formed.

The invention searches for the flanking sequence of the autosomal plateau adaptive SNP locus through a thousand human genome Project database website (1000Genomes Project, https:// www.ncbi.nlm.nih.gov/variation/tools/1000 Genomes), designs a composite amplification primer by using PrimerPremier6.0, combines with a pre-experiment result, and obtains 14 pairs of composite PCR amplification primers listed in Table 2 through repeated screening and optimization. All PCR amplification primers themselves, between the primers and the template, and between the primers, no obvious mismatch, hairpin structure and dimer structure are formed.

The kit of the invention utilizes the composite PCR amplification primer to obtain PCR amplification products of 14 autosomal plateau adaptive SNP genetic markers, and then takes the amplification products as a template to carry out single-base extension reaction. The single base extension reaction technology, also called micro sequencing technology, is a specific primer extension reaction based on four fluorescein labeled dideoxyribonucleotide. The multiple single base extension reaction can simultaneously obtain single base extension products of a plurality of SNP sites in one reaction system through a plurality of sequencing primers, and realizes one-time simultaneous detection of the plurality of SNP sites. It is characterized in that: designing single base extension primers with different lengths, and analyzing a plurality of SNP sites simultaneously, namely distinguishing different SNP sites according to different lengths of single base extension reaction products; secondly, distinguishing different alleles of SNP according to different fluorescein marked by dideoxyribonucleotide. In order to realize the simultaneous single-base extension reaction of a plurality of SNP sites, the design of a multiple single-base extension primer is a technical key, and the following factors are noted: annealing temperatures need to be approximately the same; secondly, if the bases extended by the single base are different, the SNP sites can be distinguished according to the color, if the colors are the same, 5' tailing is needed, the length of the primer is adjusted, and the single base extension products of different SNP sites are distinguished according to the electrophoretic mobility of DNA fragments with different lengths; and thirdly, no obvious mismatching, hairpin structure and dimer structure exist among the primers, the primers and the template.

In the kit, the designed single-base extension primer comprises two parts, wherein the first part is a specific sequence at the 3' end and can be complementarily combined with the SNP upstream sequence; the second part is a tail sequence at the 5' end, and tail is carried out according to the principle of GCCTCC (TCCCC) n. The added tail sequence can lead the length of the single base extension primer of different SNP sites to be different, and finally lead the length difference between the single base extension products of different sites to exist. Considering the difference in mobility of DNA fragments of different lengths in capillary electrophoresis, we designed single base extension primers for SNP loci according to the following principle: the SNP loci with the same allele are at least 5bp apart from 40bp below; 41-60bp are separated by at least 4 bp; the interval between 61 bp and 80bp is at least 3 bp; 80-87bp are separated by at least 2 bp.

It should be noted that, in order to ensure the quality of the extension primer, a reverse sequencing primer (R, reverse) is designed in a part of the site in the kit, such as the rs115321619 site. In this case, the rs115321619 site mutation type is G/A, and the actually detected allele will be the complementary base of the mutation type, i.e., C/T. When allele C is detected, the rs115321619 site allele is G; when allele T is detected, the rs115321619 site allele is A. The sites for designing reverse sequencing primers in 14 autosomal plateau adaptive SNP sites comprise rs115321619, rs73926265, rs149306391, rs374487821, rs372272284, rs150877473 and rs 116611511. Attention needs to be paid to the direction of single-base extension of the primer when analyzing the detection result.

The sequences of all 14 designed autosomal plateau adapted SNP locus composite PCR amplification primers and multiplex single-base extension primers are shown in Table 5 (the sequence numbers of each PCR amplification primer and the sequence number of the multiplex single-base extension primer are shown in tables 2 and 3, respectively).

Table 514 autosomal plateau adaptive SNP site composite amplification primers and multiple single-base extension primer reference table

In the above table, the sequence GGCCTCC (TCCCC) n before "-" represents the tailing sequence.

The kit of the invention introduces positive control as quality control and is used for accurately analyzing the genotype of a sample. The positive control provided by the invention comprises standard typing results of all 14 autosomal plateau adaptive SNP genetic markers of 2800M standard DNA. When the unknown sample is subjected to capillary electrophoresis analysis, the accuracy and reliability of the current detection result are evaluated by comparing the typing results of 2800M standard DNA.

The result detection of the kit of the invention is only carried out on a capillary electrophoresis platform. Capillary electrophoresis has been widely used in forensic genetics laboratories. The invention selects multiplex amplification and multiple single base extension technology to detect the Y chromosome SNP genetic marker, distinguishes SNP loci with the same base according to the length difference of single base extension products, and distinguishes different alleles of SNP according to the color difference of fluorescent dye. Therefore, the kit can be directly applied to any forensic genetics laboratory with a capillary electrophoresis platform, has universality and is easy to popularize.

More specifically, the kit of the invention specifically comprises the following components:

a) complex amplification reaction mixture: containing PCR buffer solution, MgCl₂Commonly used components such as dNTPs and DNA polymerase.

b) Composite amplification primer mixture: a composite amplification primer mixture composed of PCR amplification primer pairs based on 14 autosomal plateau adaptive SNP genetic markers as shown in Table 2; the composite amplification reaction mixture and the composite amplification primer mixture are used for obtaining the corresponding DNA fragments of the 14 autosomal plateau adaptive SNP genetic markers.

c) Amplification product purification reagents: comprises exonuclease 1(ExoI), Shrimp Alkaline Phosphatase (SAP); the product of the multiplex amplification is used for purification so as to be convenient for the next operation.

d) Multiplex single base extension reaction primer mix: a mixture of 14 autosomal plateau adaptive SNP genetic marker multiplex single-base extension reaction primers as described in Table 3.

e) Single base extension reaction mixture: comprises DNA polymerase, MgCl₂Buffer solution, fluorescent-labeled dideoxyribonucleic acid and the like.

The complex amplification reaction mixture, the amplification product purification reagent, and the single base extension reaction mixture may be commercially available products.

As for the extraction of the template DNA from the sample to be tested, various conventional reagents in the art can be used, and the extraction of the DNA template can be performed by referring to the conventional biochemical or genetic methods.

By using the kit provided by the invention, forensic DNA samples can be analyzed. The analysis method comprises the following steps;

1) and extracting DNA of a sample to be detected as an amplification template.

2) And (3) carrying out two-system composite amplification on the DNA extracted in the step (1) by using the composite amplification primer mixture and the composite amplification reaction mixed solution. The cycle parameters of the reaction of the multiplex amplification PCR are as follows: 15 minutes at 95 ℃; Touchdown-PCR (Touchdown-PCR) for 12 cycles at 94 ℃, 30 seconds, 66 ℃ to 60 ℃, 90 seconds, 72 ℃, 30 seconds; 94 ℃, 30 seconds, 60 ℃, 90 seconds, 72 ℃, 30 seconds, 17 cycles; then, the temperature is 72 ℃ for 10 minutes; storing at 4 ℃.

3) And (3) purifying the composite amplification product in the step (2), and performing two-system multiple single-base extension reaction by using the composite amplification product as a template and a multiple single-base extension primer mixture and a single-base extension reaction mixture. The cycle parameters of the single base extension reaction are as follows: 94 ℃, 10 seconds, 50 ℃, 5 seconds, 60 ℃, 30 seconds, 26 cycles and then storage at 4 ℃.

4) And (3) purifying the product in the step (3) by using shrimp alkaline phosphatase, and then performing capillary electrophoresis analysis to obtain a genotyping result of the sample according to the electrophoresis result.

Further, the analysis of the multiple single-base extension products in step 4 of the above method comprises the following steps: firstly, analyzing the 2800M standard DNA typing result, and if the standard DNA typing result is accurate, continuing to analyze the sample to be detected to obtain the genotype of the sample to be detected.

The following examples are intended to illustrate specific embodiments of the present invention without limiting the scope of the invention to the examples.

In the examples, the following reagents and instruments were used unless otherwise specified;

1) gene analyzer 3130, ABI corporation;

2) ProFlexPCR amplification apparatus, ABI company;

3) bench high speed centrifuge, EPPENDORF corporation;

4) pure water unit, Millipore corporation;

5) pipettor, EPPENDORF;

6) Hi-Di formamide, ABI corporation;

7) exonuclease 1, takara biotechnology;

8) shrimp alkaline phosphatase, takara biotechnology;

9) internal standard (Genescan)^TMSizeStandardGS-120LIZ), ABI.

EXAMPLE 1 preparation of the kit of the invention

The 14 autosomal plateau adaptive SNP genetic marker composite detection kit for detection can comprise the following reagents packaged respectively:

a) and (3) compounding the amplification primer mixture. Amplification primers shown in Table 2 were mixed, and the amplification primers were synthesized by Invitrogen, and the synthesized amplification primers were mixed at a ratio shown in Table 6 with ultrapure water of 100 pM/. mu.l.

b) And (3) mixing the amplification reaction solution. In this example, a PCR reaction mixture Multiplex PCRMix from Qiagen was used.

c) Multiplex single base extension reaction primer mix. The primers were obtained by mixing the single-base extension primers shown in Table 3, and were synthesized by Invitrogen corporation. The synthesized 14 extension primers (50 pM/. mu.l with ultrapure water) were used to prepare a multiplex single-base extension primer mixture according to the parameters given in Table 7.

d) A single base extension reaction mixture. In this example, a SNaPshot ready interaction mix, a product of ABI, was used.

e) Standard DNA2800M, ABI Standard DNA2800M was used in this example.

The reagents are respectively packaged according to respective conventional requirements to prepare the composite detection kit based on 14 autosomal plateau adaptive SNP genetic markers for subsequent experiments.

TABLE 6 concentration of composite PCR amplification primers

TABLE 7 concentration of multiplex single base extension primers

Serial number	SNP loci	Direction of extension of primer	Extended primer Length (bp)	Primer concentration (μ M)
					1	rs115321619	R		30	0.221
2	rs13419896	F		35							0.111
					3	rs73926265	R		40	0.111
4	rs55981512	F	44	0.111
					5	rs116611511	R	48	0.111
6	rs189807021	F	52	0.111
					7	rs372272284	R		55	0.111
8	rs374487821	R	59	0.111
					9	rs73926264	F	63	0.111
10	rs150877473	R	67	0.166
					11	rs73926263	F	71	0.044
12	rs149306391	R	75	0.066
					13	rs369097672	F	79	0.331
14	rs142826801	F	82	0.089

Example 2 200 samples of a population of high-altitude humans were tested using the kit of the invention.

The forensic medicine composite detection kit based on 14 autosomal plateau adaptive SNP genetic markers is used for detecting 200 plateau population samples, wherein the plateau population samples are from an Ali, a Japanese karst, a Naqu and a southern mountain area with the average altitude of more than 2500 m. The specific detection process is carried out according to the following steps:

a. extracting DNA of 200 plateau population samples by using a Chelex-100 method to serve as a composite amplification template;

b. b, taking the DNA in the step a as a template, and carrying out composite PCR amplification on the sample in an amplification system by using a composite amplification primer mixture and a composite amplification reaction mixed solution; mu.l of the composite amplification primer mixture, 2.5. mu.l of the composite amplification reaction mixture, 0.8. mu.l of the template DNA, and ddH₂O to 5 μ l; 15 minutes at 95 ℃; Touchdown-PCR (Touchdown-PCR) for 12 cycles at 94 ℃, 30 seconds, 66 ℃ to 60 ℃, 90 seconds, 72 ℃, 30 seconds; 94 ℃, 30 seconds, 60 ℃, 90 seconds, 72 ℃, 30 seconds, 17 cycles; then, the temperature is 72 ℃ for 10 minutes; storing at 4 ℃.

c. Purifying a multiplex PCR product; purification system of amplification product for each sample: ExoI (5U/. mu.l) 0.8. mu.l, SAP (1U/. mu.l) 2.5. mu.l, multiplex PCR product 5. mu.l; amplification product purification reaction conditions: 60 minutes at 37 ℃ and 10 minutes at 80 ℃; storing at 4 ℃.

d. Taking the product obtained by the purification in the previous step as a template, and carrying out single base extension reaction by utilizing a multiple single base extension primer mixture; the method comprises the following steps: 1.5. mu.l of single base extension reaction mixture, 0.18. mu.l of multiplex single base extension primer mixture, 1.5. mu.l of purified amplification product, and ddH₂O to 5 μ l; thermal cycling parameters for single base extension reactions: circulating for 25 times at 96 ℃ for 10 seconds, 50 ℃ for 5 seconds and 60 ℃ for 30 seconds; storing at 4 ℃.

e. Purifying the single base extension reaction product in the last step; and (3) purification system: SAP (1U/. mu.l) 1. mu.l, single base extension reaction product 5. mu.l; purification reaction conditions: storing at 37 deg.C for 60 min, 80 deg.C for 10 min, and 4 deg.C;

f. capillary electrophoresis:

respectively taking 1 mu l of the purified extension product obtained in the step e, adding 10 mu l of Hi-Di methanolamine and 0.1 mu l of internal standard, and uniformly mixing; then, the mixture was denatured at 95 ℃ for 3 minutes, rapidly cooled at 4 ℃ and subjected to electrophoresis using a Gene Analyzer 3130 manufactured by ABI, USA. Electrophoresis conditions: 1500V voltage, 36cm capillary, POP7 gel, electrophoresis for 18 minutes; data Collection3.0 software was used to collect Data, and GeneMapper ID V3.2 software was used to perform the results analysis.

The results for 200 plateau population samples are shown in table 8 and fig. 1.

TABLE 8 test results

FIG. 1b shows the results of typing standard DNA2800, and a and c show the results of typing a plateau population sample. It can be seen that both the plateau population and the standard DNA can clearly distinguish the alleles of all 14 plateau adaptive SNP sites. The typing results of 200 plateau samples collected in the Ali, Japanese karya, Naqu and southern mountain areas with altitude exceeding 3500 m show that the mutation frequency of 6 or more SNP sites adapted to 14 plateaus in the plateau population is 97% (194/200), and the mutation frequency of 6 or more SNP sites in the Han nationality population of the 14 sites in the thousand-people genome planning database is only 0.96% (2/208). The mutation of 6 or more SNP sites adapted to 14 plateau regions is set as a test standard, and when the mutation of 6 or more SNP sites adapted to 14 plateau regions occurs in an unknown sample, the ratio of the probability that the sample belongs to the plateau region population to the probability that the sample belongs to the low-altitude region population is 101.04. Therefore, the forensic composite detection kit based on 14 autosomal plateau adaptive SNP genetic markers has strong sensitivity and specificity for distinguishing plateau population and low-altitude population, and can be applied to forensic actual detection cases.

Sequence listing

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acaggcatta ggcatgtgag tc 22

<210> 6

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gaatctaagg atgatctgct cttggt 26

<210> 7

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cacctgcggc atagatagat actg 24

<210> 8

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gtaggactaa gaaccagcat cagag 25

<210> 9

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gagtaagagc cctgaatttg gtttg 25

<210> 10

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tctcccttta tctgtgccac tga 23

<210> 11

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ggtagaaatg agtggaaggt gcta 24

<210> 12

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cttggtctgt taatgagtca gtagaga 27

<210> 13

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gcatggcttg ttggagctac tt 22

<210> 14

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tcctctcatc tactggcacc tg 22

<210> 15

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ttgtcagagc attgactcca gatt 24

<210> 16

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gcacaggcta tggcattaac g 21

<210> 17

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

aaattgagac ccacggctct g 21

<210> 18

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

atgcaaacag ctatgtcacc tttc 24

<210> 19

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

cactctcggc tccatgtctg a 21

<210> 20

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gatgatgagg caggacagca g 21

<210> 21

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

accttctgtg tggcatctat ttca 24

<210> 22

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

actgtgtcca tattcactgt gtcc 24

<210> 23

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

aagccactat ctcctacagt taagc 25

<210> 24

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

ccaagtgagg taggcaagac ag 22

<210> 25

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

ctgtgaagca aatcataacc ctctg 25

<210> 26

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

gctggcttac ctgtaaagta tatgttt 27

<210> 27

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

aaggtgtgga actgaacata gacg 24

<210> 28

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

accctgaggc tctgtcaatg g 21

<210> 29

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

gcctcctccc ctgccatgca ccactgcact 30

<210> 30

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

gcctcctccc ctcggccagt gtctgaaagt gaagc 35

<210> 31

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

gcctcctccc ctcccctccc ccacagcctg gagtgaggcc 40

<210> 32

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

gcctcctccc ctcccctccc ctcccctccc cttcttcccc cgcc 44

<210> 33

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

gcctcctccc ctcccctccc ctcccctccg ccactgaggc tcctctgc 48

<210> 34

<211> 52

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

gcctcctccc ctcccctccc ctcccctccc ctccgctgac ctcaggccac ac 52

<210> 35

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

gcctcctccc ctcccctccc ctcccttttt aaacctcttc tccatcaaaa cctac 55

<210> 36

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

gcctcctccc ctcccctccc ctcccctccc ctcccctccc cttccctctg ccattggcc 59

<210> 37

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

gcctcctccc ctcccctccc ctcccctccc ctcccctccc ctttattaaa tgcatctggc 60

ccc 63

<210> 38

<211> 67

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

gcctcctccc ctcccctccc ctcccctccc ctcccctccc ctcccctccg tgcagtgcaa 60

gacctga 67

<210> 39

<211> 71

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

gcctcctccc ctcccctccc ctcccctccc ctcccctccc ctcctctcat tattttcatc 60

tccctcttcc t 71

<210> 40

<211> 75

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

gcctcctccc ctcccctccc ctcccctccc ctcccctccc ctcccctcct ttcacagatc 60

tggaaacaga cactt 75

<210> 41

<211> 79

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

gcctcctccc ctcccctccc ctcccctccc ctcccctccc ctcccctccc ttagacaaga 60

tgatacaacc attttggaa 79

<210> 42

<211> 82

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

gcctcctccc ctcccctccc ctcccctccc ctcccctccc ctcccctccc ctccttaaag 60

gtgtggaact gaacatagac gt 82

Claims

1. A forensic medicine composite detection kit based on 14 autosomal SNP genetic markers is characterized in that: the composition comprises: separating the packaged composite amplification primer mixture, the multiple single-base extension reaction primer mixture and the standard DNA; the number of the composite amplification primer mixtures is 28; the primer mixture of the multiple single-base extension reaction has 14 pieces;

the 14 autosomal SNP sites are shown below: rs115321619, rs13419896, rs73926265, rs55981512, rs116611511, rs189807021, rs372272284, rs374487821, rs73926264, rs150877473, rs73926263, rs149306391, rs369097672, rs 142826801;

the nucleotide sequence of each amplification primer in the composite amplification primer mixture is shown as SEQ ID No.1-SEQ ID No. 28;

the nucleotide sequence of each primer in the multiple single-base extension reaction primer mixture is shown as SEQ ID NO.29-SEQ ID NO. 42.

2. The forensic medicine composite detection kit based on 14 autosomal SNP genetic markers according to claim 1, wherein: the standard DNA was 2800M standard DNA.

3. The forensic medicine composite detection kit based on 14 autosomal SNP genetic markers according to claim 1, wherein: the composition also includes: composite amplification reaction mixed liquor, amplification product purification reagent and single base extension reaction mixed liquor; the composite amplification reaction mixed solution is a PCR reaction mixed solution Multiplex PCR Mix of Qiagen company, the amplification product purification reagent is a reagent containing exonuclease 1 and shrimp alkaline phosphatase, and the single-base extension reaction mixed solution is a SNaPshot ready reaction Mix of ABI company.

4. Use of the forensic medicine composite detection kit based on 14 autosomal SNP genetic markers as defined in any one of claims 1 to 3 for differentiating between a high-altitude population and a low-altitude population.

5. The use method of the forensic medicine composite detection kit based on 14 autosomal SNP genetic markers according to any one of claims 1 to 3, comprising the steps of:

a. extracting DNA of a sample to be detected as an amplification template;

b. b, performing two-system composite amplification on the DNA extracted in the step a by adopting a composite amplification primer mixture and a composite amplification reaction mixed solution; the composite amplification reaction mixed solution is a PCR reaction mixed solution Multiplex PCR Mix of Qiagen company;

c. purifying the composite amplification product in the step b, and performing multiplex single-base extension reaction of two systems by using the composite amplification product as a template and using a multiplex single-base extension primer mixture and a single-base extension reaction mixed solution; the mixed solution of the single base extension reaction is a product SNaPshot ready reaction mix of ABI company;

d. and (c) purifying the product obtained in the step (c) by using shrimp alkaline phosphatase, and performing capillary electrophoresis analysis to obtain a genotyping result of the sample according to the electrophoresis result.

6. Use according to claim 5, characterized in that: the cycle parameters of the reaction of the multiplex amplification PCR in the step b are as follows: 15 minutes at 95 ℃; 12 cycles of touchdown amplification at 94 ℃, 30 seconds, 66 ℃ to 60 ℃, 90 seconds, 72 ℃, 30 seconds; 17 cycles of 94 ℃, 30 seconds, 60 ℃, 90 seconds, 72 ℃, 30 seconds, then 72 ℃, 10 minutes; storing at 4 ℃.

7. Use according to claim 6, characterized in that: the cycle parameters of the single base extension reaction in the step c are as follows: 94 ℃, 10 seconds, 50 ℃, 5 seconds, 60 ℃, 30 seconds, 26 cycles and then storage at 4 ℃.