Composite amplification detection system for DIP polymorphic sites of X chromosome and application of composite amplification detection system

Abstract

The invention discloses a composite amplification detection system of X chromosome DIP polymorphic loci and application thereof, the composite amplification detection system comprises 50 X chromosome DIP polymorphic loci, a sex identification locus Amelogenin and two Y chromosome DIP loci, 15 loci of the 50 X chromosome DIP polymorphic loci can form 6 linkage groups, and 15 loci of the 50 X chromosome DIP polymorphic loci can form 6 linkage groups. And the method has higher forensic medicine application efficiency. According to the multiplex amplification detection system disclosed by the invention, a miniaturized amplicon (70-205 bp) is designed, so that accurate typing analysis on a degraded detection material in forensic practice is effectively carried out. Meanwhile, the multiplex amplification detection system is simple, convenient, low in cost and easy to popularize, and has important significance on individual identification and complex genetic relationship identification of East Asian population.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gene detection, and particularly relates to a composite amplification detection system for X chromosome DIP polymorphic sites and application thereof. BACKGROUND

[0002] As an important sex chromosome of human, X chromosome has irreplaceable application value in forensic kinship identification due to its unique genetic pattern. The full length of X chromosome is about 155 Mb, containing about 1100 genes, accounting for 5% of the coding genes in human genome. In terms of genetic characteristics, female individuals carry two X chromosomes of paternal and maternal origins, which can undergo homologous recombination and be randomly inherited to offspring in the process of meiosis; while male individuals only carry a single maternal X chromosome, except for the pseudo-autosomal region, and lack of homologous recombination with Y chromosome, and are inherited to daughters in the form of haplotype. This sex-linked genetic characteristic makes X chromosome show special advantages in maternal-granddaughter, half-sister of the same father and complex kinship identification involving incest: 1. Without considering mutation, the grandmother and granddaughter have one same allele at each X chromosome genetic marker; 2. The half-sisters of the same father share the same paternal X chromosome; 3. In the identification of parentage involving incest, if two disputed fathers are father-son relationship, they can be regarded as unrelated individuals for comparison. Studies have shown that in complex kinship identification such as grandchild identification, when autosomal markers cannot provide a clear conclusion, X chromosome markers often provide key supplementary evidence, highlighting their important supplementary identification value.

[0003] Insertion / deletion polymorphism (DIP), as a special genetic marker, is manifested as the insertion or deletion of DNA fragments of different lengths in human genome. DIP has the advantages of both short tandem repeat (STR) and single nucleotide polymorphism (SNP), and is the most abundant type of DNA polymorphism after SNP, mainly showing biallelic. Compared with SNP, DIP belongs to length polymorphism, which is suitable for the capillary electrophoresis technology platform popularized in existing forensic DNA laboratories, and has the advantages of simple typing, easy operation and strong popularization. Compared with STR, the mutation rate of DIP is lower, which is comparable to SNP, about 10 -8Thus, it is more stable. In addition, the di-allele structure of DIP makes the alleles known and fixed, and the amplicon can be designed as a short fragment, suitable for degraded DNA sample detection. The main shortcomings of existing research are as follows: 1. The polymorphism of part of X-DIP sites in the East Asian population is poor; 2. The existing X-DIP system has limited site capacity (more than 40); 3. The X-DIP sites located in the linkage group are not fully excavated; 4. The amplicon fragment of part of the system is too long, which is not conducive to the accurate typing detection of degraded samples; 5. The identification efficiency of complex kinship still needs to be improved.

[0004] In view of the shortcomings of the existing X-DIP research, the present application provides a composite amplification detection system of X chromosome DIP polymorphic sites and its application, mainly based on screening single X-DIP sites with high polymorphism in the East Asian population, and integrating X-DIP sites in linkage groups, further improving the overall polymorphism of the system, and designing short amplicons (about 200bp) to better adapt to the accurate typing of trace degraded samples. SUMMARY

[0005] The present application aims to at least solve one of the above technical problems in the prior art. To this end, the purpose of the present application is to provide a composite amplification detection system of X chromosome DIP polymorphic sites and its application.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is: In a first aspect of the present application, an X chromosome DIP locus combination is provided, which comprises 50 X chromosome DIP loci, 2 Y chromosome DIP loci and an Amelogenin identification locus, the 50 X chromosome DIP loci being: rs3052378, rs35225800, rs56971671, rs3831664, rs10600022, rs3050111, rs34023916, rs72407549, rs150915048, rs4017281, rs34764424, rs10569568, rs35543200, rs201126330, rs143246211, rs56238119, rs145745343, rs72422496, rs200046032, rs58459443, rs10669433, rs34179015, rs33920981, rs55921940, rs3831660, rs35417817, rs144674109, rs2308239, rs10533508, rs72566727, rs199731653, rs35843349, rs67315811, rs35244442, rs36094418, rs60501705, rs3859989, rs60477615, rs59550361, rs58470327, rs35358831, rs5902767, rs143535638, rs10695276, rs10638624, rs10549472, rs77572119, rs199644583, rs61260787, rs72384910, the two Y chromosome DIP loci being rs759551978 and rs2032678 respectively.

[0007] In a second aspect of the present application, the reagent for detecting the X chromosome DIP locus combination is applied in parentage identification and individual identification.

[0008] In some embodiments of the present application, the reagent comprises a primer pair for amplifying the X chromosome DIP locus molecular marker combination.

[0009] In some embodiments of the present application, the sequence of the primer pair is shown in SEQ ID NO: 1-100.

[0010] In some embodiments of the present application, the concentration of the primer pair is 0.05-1 μM.

[0011] In some embodiments of the application, the concentration of the pair of primers is 0.06-0.9 μM.

[0012] In some embodiments of the application, the concentrations of the sequences of the primers are as follows: the primer pair of SEQ ID NO: 1-2 is at a concentration of 0.2351 μΜ; the primer pair of SEQ ID NO: 3-4 is at a concentration of 0.1754 μΜ; the primer pair of SEQ ID NO: 5-6 is at a concentration of 0.2277 μΜ; the primer pair of SEQ ID NO: 7-8 is at a concentration of 0.1064 μΜ; the primer pair of SEQ ID NO: 9-10 is at a concentration of 0.1568 μΜ; the primer pair of SEQ ID NO: 11-12 is at a concentration of 0.2426 μΜ; the primer pair of SEQ ID NO: 13-14 is at a concentration of 0.1829 μΜ; the primer pair of SEQ ID NO: 15-16 is at a concentration of 0.2519 μΜ; the primer pair of SEQ ID NO: 17-18 is at a concentration of 0.1493 μΜ; the primer pair of SEQ ID NO: 19-20 is at a concentration of 0.1866 μΜ; the primer pair of SEQ ID NO: 21-22 is at a concentration of 0.1735 μΜ; the primer pair of SEQ ID NO: 23-24 is at a concentration of 0.3676 μΜ; the primer pair of SEQ ID NO: 25-26 is at a concentration of 0.2202 μΜ; the primer pair of SEQ ID NO: 27-28 is at a concentration of 0.3396 μΜ; the primer pair of SEQ ID NO: 29-30 is at a concentration of 0.1978 μΜ; the primer pair of SEQ ID NO: 31-32 is at a concentration of 0.2855 μΜ; the primer pair of SEQ ID NO: 33-34 is at a concentration of 0.3004 μΜ; the primer pair of SEQ ID NO: 35-36 is at a concentration of 0.1922 μΜ; the primer pair of SEQ ID NO: 37-38 is at a concentration of 0.2146 μΜ; the primer pair of SEQ ID NO: 39-40 is at a concentration of 0.3658 μΜ; the primer pair of SEQ ID NO: 41-42 is at a concentration of 0.3060 μΜ; the primer pair of SEQ ID NO: 43-44 is at a concentration of 0.2892 μΜ; the primer pair of SEQ ID NO: 45-46 is at a concentration of 0.2426 μΜ; the primer pair of SEQ ID NO: 47-48 is at a concentration of 0.2799 μΜ; the primer pair of SEQ ID NO: 49-50 is at a concentration of 0.2482 μΜ; the primer pair of SEQ ID NO: 51-52 is at a concentration of 0.2239 μΜ; the primer pair of SEQ ID NO: 53-54 is at a concentration of 0.2109 μΜ; the primer pair of SEQ ID NO: 55-56 is at a concentration of 0.2127 μΜ; the primer pair of SEQ ID NO: 57-58 is at a concentration of 0.4217 μΜ; the primer pair of SEQ ID NO: 59-60 is at a concentration of 0.3359 μΜ; the primer pair of SEQ ID NO: 61-62 is at a concentration of 0.3770 μΜ; the primer pair of SEQ ID NO: 63-64 has a concentration of 0.2202 μΜ; the primer pair of SEQ ID NO: 65-66 has a concentration of 0.4908 μΜ; the primer pair of SEQ ID NO: 67-68 has a concentration of 0.3508 μΜ; the primer pair of SEQ ID NO: 69-70 has a concentration of 0.2874 μΜ; the primer pair of SEQ ID NO: 71-72 has a concentration of 0.4180 μΜ; the primer pair of SEQ ID NO: 73-74 has a concentration of 0.2501 μΜ; the primer pair of SEQ ID NO: 75-76 has a concentration of 0.4684 μΜ; the primer pair of SEQ ID NO: 77-78 has a concentration of 0.3937 μΜ; the primer pair of SEQ ID NO: 79-80 has a concentration of 0.3695 μΜ; the primer pair of SEQ ID NO: 81-82 has a concentration of 0.4647 μΜ; the primer pair of SEQ ID NO: 83-84 has a concentration of 0.3919 μΜ; the primer pair of SEQ ID NO: 85-86 has a concentration of 0.3134 μΜ; the primer pair of SEQ ID NO: 87-88 has a concentration of 0.3042 μΜ; the primer pair of SEQ ID NO: 89-90 has a concentration of 0.5953 μΜ; the primer pair of SEQ ID NO: 91-92 has a concentration of 0.3266 μΜ; the primer pair of SEQ ID NO: 93-94 has a concentration of 0.5113 μΜ; the primer pair of SEQ ID NO: 95-96 has a concentration of 0.5225 μΜ; the primer pair of SEQ ID NO: 97-98 has a concentration of 0.3191 μΜ; the primer pair of SEQ ID NO: 99-100 has a concentration of 0.8724 μΜ.

[0013] In some embodiments of the present application, at least one primer in the primer pair is labeled with a fluorescent dye at the 5' end.

[0014] In some embodiments of the present application, the fluorescent dye comprises FAM, HEX, SUM, TAMRA, LYN, ROX, ATTO or PUR.

[0015] In some embodiments of the present application, the primer pair is labeled with different fluorescent dyes, respectively.

[0016] In some embodiments of the present application, the primer pairs of SEQ ID NO: 1-24, SEQ ID NO: 25-44, SEQ ID NO: 45-66, SEQ ID NO: 67-84 and SEQ ID NO: 85-100 are labeled with different fluorescent dyes, respectively.

[0017] In some embodiments of the present application, the primer pairs of SEQ ID NO: 1-24 are labeled with FAM, the primer pairs of SEQ ID NO: 25-44 are labeled with HEX, the primer pairs of SEQ ID NO: 45-66 are labeled with TAMRA, the primer pairs of SEQ ID NO: 67-84 are labeled with ROX, and the primer pairs of SEQ ID NO: 85-100 are labeled with ATTO.

[0018] In some embodiments of the present application, the sequence fragments targeted for amplification by the primer pairs are less than 210 bp in length.

[0019] In a third aspect of the present application, the use of the X chromosome DIP site combination described above in the preparation of a kit, wherein the kit comprises an allelic typing standard prepared based on the X chromosome DIP site described above.

[0020] In some embodiments of the present application, the allelic typing standard is an amplification product obtained by separately amplifying the 50 X chromosome DIP sites described above.

[0021] In some embodiments of the present application, the amplification product is a heterozygous amplification product.

[0022] In some embodiments of the present application, the allelic typing standard comprises all alleles of the X chromosome DIP sites described above.

[0023] In a fourth aspect of the present application, a method for detecting genetic polymorphisms of human X chromosome DIP sites is provided, characterized in that the method comprises the following steps: using the reagent or kit described above to perform PCR amplification on a sample, and performing genotyping analysis on the PCR amplification product to obtain the genetic polymorphisms of the human X chromosome DIP sites.

[0024] In some embodiments of the present application, the amplification system is:

[0025] In some embodiments of the present application, the amplification procedure is:

[0026] In some embodiments of the present application, the method for performing genotyping analysis on the PCR amplification product comprises capillary electrophoresis or polyacrylamide gel electrophoresis.

[0027] The beneficial effects of the present application are: The application provides a composite amplification detection system for X chromosome DIP polymorphic sites and application thereof. 50 X-DIP polymorphic sites selected in the application can form 6 linkage groups, and 15 sites have higher polymorphism and higher forensic application efficiency compared with single X-DIP sites, and are more suitable for forensic individual identification and complex kinship identification and are more suitable for individual identification and complex kinship identification of East Asian populations.

[0028] The composite amplification detection system for X-DIP polymorphic sites provided in the application can effectively analyze the accurate typing of degraded samples in forensic practice by designing miniaturized amplicons (70-205 bp), thereby providing technical support for public security science and technology and providing scientific basis for court trials. The efficiency of the composite amplification detection system in the diad paternity test is higher than 0.9999, and the system can play an auxiliary role in complex kinship identification. The application constructs a simple, low-cost and easy-to-promote composite amplification detection system suitable for forensic practice, solves the problems of insufficient site capacity and limited polymorphism information of the existing X-DIP detection system, and has important significance for assisting forensic individual identification and complex kinship identification. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is a fluorescence labeling type, amplicon size and site arrangement diagram of 50 X-DIP sites, tooth enamel loci (Amelogenin) and 2 Y-DIP sites in the application.

[0030] Figure 2 It is a capillary electrophoresis detection spectrum of the allelic typing standard prepared in the embodiment of the application.

[0031] Figure 3 It is a typing spectrum of the DNA standard 9948 detected by the composite amplification system of the application.

[0032] Figure 4 It is a typing spectrum of the male human DNA sample detected by the composite amplification system of the application.

[0033] Figure 5 It is a typing spectrum of the female human DNA sample detected by the composite amplification system of the application.

[0034] Figure 6 It is a sensitivity detection result spectrum of the composite amplification system of the application.

[0035] Figure 7 It is a typing spectrum of the saliva swab sample stored for 11 years detected by the composite amplification system of the application. DETAILED DESCRIPTION

[0036] The present application will be further described in details by specific examples. The raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial channels or can be obtained by prior art methods, unless otherwise specified. The tests are conventional methods in the art, unless otherwise specified.

[0037] Example 1 Screening of X chromosome DIP polymorphic sites Based on the X chromosome genomic data of 2504 individuals in the 1000 Genomes Project phase 3 database, a multi-stage screening strategy was used to obtain highly polymorphic X chromosome DIP (X-DIP) sites. The specific screening criteria include: 1. Located in the intronic region or intergenic variant region of the X chromosome; 2. Di-allelic DIP polymorphism; 3. Insertion / deletion fragment length of 2bp-6bp; 4. Minor allele frequency (MAF) in East Asian population >0.3; 5. In Hardy-Weinberg equilibrium in East Asian population; 6. Loci in linkage must meet R 2 >0.2 and D' value >0.5, forming a linkage group; 7. The physical distance between non-linked loci is greater than 0.1Mb, and the loci are in linkage equilibrium (R 2 <0.2); 8. Verified by NCBI database, excluding other polymorphic sites in the flanking sequence of 200bp upstream and downstream of the target site. After the above strict screening, the final set of X-DIP candidate sites meeting the requirements is shown in Table 1.

[0038] Table 1 Basic information of 50 X-DIP sites

[0039] Among them, the rs number, reference allele, replacement allele, minor allele frequency (MAF) and physical position refer to the dbSNP database (GRCh37). In order to perform gender determination, a tooth enamel locus (Amelogenin) and 2 Y chromosome DIP (Y-DIP) sites (rs759551978 and rs2032678) are added for accurate gender identification.

[0040] Example 2 Population genetics analysis of 50 X-DIP sites Based on the data of East Asian population (n=504, 244 males and 260 females) in the 1000 Genomes Project phase 3 dataset, the allele frequency, haplotype block frequency and forensic parameters of 50 X-DIP loci were statistically analyzed by StatsX v2.0 software, and the Hardy-Weinberg equilibrium test, linkage analysis and allele frequency difference analysis between genders were performed by Arlequin software. The forensic parameters of each X-DIP locus and haplotype block, including the Power of Discrimination (PD), Cumulative Power of Discrimination (CPD), Mean Exclusion Chance (MEC) and Cumulative Mean Exclusion Chance, etc. are shown in Table 2 and Table 3 as follows.

[0041] Table 2 Forensic parameters of 50 X-DIP loci

[0042] Note: MEC_Kruger refers to the average exclusion probability calculated by the method proposed by Kruger in the absence of relatives in the identification of kinship (mother-daughter, grandmother-granddaughter) (Krüger et al., 1968); MEC_Kishida refers to the average exclusion probability calculated by the method proposed by Kishida in the standard triad paternity test (including daughter) (Kishida et al., 1997); MEC_Desmarais refers to the average exclusion probability calculated by the method proposed by Desmarais in the triad paternity test (including daughter) (Desmarais et al., 1998); MEC_Desmarais_duo refers to the average exclusion probability of two pairs calculated by the method proposed by Desmarais in the identification of parent-child (father-daughter or mother-son) (Desmarais et al., 1998); LG1: Haplotype block 1 (rs35244442-rs77572119); LG2: Haplotype block 2 (rs35843349-rs2308239); LG3: Haplotype block 3 (rs35358831-rs60477615-rs72384910); LG4: Haplotype block 4 (rs143535638-rs10695276-rs60501705); LG5: Haplotype block 5 (rs61260787-rs3859989); LG6: Haplotype block 6 (rs34764424-rs10569568-rs10669433).

[0043] Table 3 Cumulative forensic parameters of 50 X-DIP loci

[0044] The results showed that there was no statistically significant difference in allele frequency between male and female populations, and all of them met the Hardy-Weinberg equilibrium after Bonferroni correction.

[0045] Example 3 X-DIP locus primer design Based on the X-DIP candidate loci screened in Example 1, a systematic primer design process was used to design and evaluate each locus.

[0046] 1 Primer design The flanking sequence information of 200 bp upstream and downstream of the target DIP locus was obtained from the UCSC Human Genome Browser, and the primer was designed through the Primer3 online platform (https: / / primer3.ut.ee / ). The design process followed the following principles: 1. The length of the primer was controlled at 20 bp ~ 28 bp; 2. The length of the amplicon was 70 bp ~ 210 bp; 3. The annealing temperature (Tm value) of the primer was in the range of 57℃ ~ 62℃ (the optimal Tm value was 60℃), and the Tm value of the forward and reverse primers differed by no more than 3℃; 4. The GC content of the primer sequence was between 30% ~ 70%, and was preferably distributed between 40% ~ 60%; 5. The primer sequence avoided the continuous occurrence of more than 5 single base repeats, and the 3' end of the primer sequence was preferably not more than 3 consecutive C or G; 6. The specificity of the primer pair was checked using the In-Silico PCR function of the UCSC Human Genome Browser and the Primer-BLAST function of NCBI; 7. The primer dimer risk was evaluated using AutoDimer software, and the free energy score of the primer itself and the interaction between primers was required to be ≤ 8 points.

[0047] 2 Verification and evaluation of primers After unit point PCR amplification, the electrophoresis was subjected to agarose gel electrophoresis test to detect the specificity and amplification efficiency of the primers. The unit point PCR amplification system is shown in Table 4.

[0048] Table 4 Reaction components and volumes of unit point amplification system

[0049] Among them, Taq PCR premix (2x) was purchased from Shengong Bioengineering (Shanghai) Co., Ltd.

[0050] The specific PCR experiment steps are as follows: DNA was extracted from the genomic DNA extraction kit (TIANGEN BIOTECH) using an oral swab according to the instructions, eluted with 40 μL TB buffer, and the DNA concentration was determined using the Qubit dsDNA HS Assay Kit (ThermoFisher). After preparing the PCR reaction system as described above, the amplification was carried out on an ABI 9700 type PCR amplification instrument according to the following reaction program: (1) 95°C initial denaturation for 2 minutes, 1 cycle; (2) 94°C denaturation for 30 seconds, 60°C annealing for 1 minute, 72°C extension for 50 seconds, 31 cycles; (3) 60°C final extension for 60 minutes, 1 cycle.

[0051] The amplified products were analyzed by 2% agarose gel electrophoresis. The performance of the primers was evaluated by agarose electrophoresis results: when a single, bright and narrow band was observed, it indicated that the primers had good specificity and amplification efficiency; if the band signal was weak, it suggested that the PCR conditions needed to be optimized, including adjusting the annealing temperature or the number of cycles; when multiple non-specific bands were detected, it was determined that the primers were not specific enough and needed to be redesigned.

[0052] The 5' end of the upstream or downstream primer of the primer tested by agarose gel electrophoresis was modified with fluorescence, and the specificity of the primer and whether the length of the amplified fragment was consistent with the expected design were further verified on a capillary electrophoresis platform. The specific experimental steps are as follows: (1) PCR was performed according to the above PCR system and reaction program; (2) The PCR product obtained in (1) was mixed and centrifuged, 1 μL of the product was mixed with 0.5 μL AGCU Marker SIZ-500 internal standard and 9.5 μL deionized formamide, and added to a 96-well plate, centrifuged at 2000 rpm for 3 min, denatured at 95°C for 3 min, and then ice bathed for 3 min; (3) Then, the 96-well plate after ice bath in (2) was placed on a 31300xL gene analyzer for capillary electrophoresis separation. After electrophoresis, the GeneMapper® ID-X v1.7 (Applied Biosystems, USA) software was used for genotyping analysis. Based on the single-site amplification results, a Bin file was established for each site, in which the insertion allele of the X-DIP site was named I, and the deletion allele was named D. Then the Bin files of the 50 X-DIP sites were integrated into a Panel file, named 50-Plex X-DIP, and the file was imported into the analysis method of the genotyping software GeneMapper to realize automatic typing.

[0053] The screening criteria for capillary electrophoresis results are that only single peaks are amplified in male samples, while in female samples, homozygous single peaks and heterozygous double peaks may appear, and the balance of the peak height of the heterozygous double peaks is good. The primer pairs that meet the conditions are included in the subsequent experiments.

[0054] Based on the above principles and methods, 50 X-DIP sites, Amelogenin and 2 Y chromosome DIP (Y-DIP) site primer sequences were obtained. The primer sequence information is shown in Tables 5-9, and the site arrangement diagram is shown in Figure 1 All primers are grouped according to the expected fragment length (70-205 bp) (as shown in Figure 1 The first group of X-DIP site primers (SEQ ID NO. 1-2 to SEQ ID NO. 23-24) are labeled with 6-FAM fluorescent dye, the second group of X-DIP site primers (SEQ ID NO. 25-26 to SEQ ID NO. 43-44) are labeled with HEX fluorescent dye, the third group of X-DIP site primers (SEQ ID NO. 45-46 to SEQ ID NO. 65-66) are labeled with TAMRA fluorescent dye, the fourth group of X-DIP site primers (SEQ ID NO. 67-68 to SEQ ID NO. 83-84) are labeled with ROX fluorescent dye, and the fifth group of X-DIP site primers (SEQ ID NO. 85-86 to SEQ ID NO. 99-100) are labeled with ATTO fluorescent dye. The primer sequences of Y-DIP sites (rs759551978h and rs2032678) (SEQ ID NO. 104-105 and SEQ ID NO. 106-107) are labeled with ROX fluorescent dye, and the 5' end of the upstream primer in all primer pairs is labeled with fluorescent dye. The primer sequence of the Amelogenin locus (Amelogenin) (SEQ ID NO. 100-103) is labeled with ATTO fluorescent dye.

[0055] At the same time, due to the significant differences in the fragment size of the amplification products of each X-DIP site and the amplification efficiency of the primers, based on the amplification detection results of a single X-DIP site, new sites are introduced into the amplification system for testing using the step-by-step superposition method, that is, on the basis of single-site amplification system, gradually add primer pairs of new sites, evaluate the specificity of amplification and whether there is related influence between primers through capillary electrophoresis typing results, verify and screen all X-DIP site primer pairs system, optimize and adjust the primer concentration of each site through a series of experiments, gradually improve the amplification balance of primer combinations, and finally obtain the optimal concentration of each primer pair in the amplification composite detection system as shown in Tables 5-9.

[0056] Table 5 Primer sequence information table of FAM-labeled X-DIP site

[0057] Table 6 Primer sequence information table of HEX-labeled X-DIP site

[0058] Table 7 Primer sequence information table of HEX-labeled X-DIP site

[0059] Table 8 Primer sequence information table of ROX-labeled X-DIP site and two Y chromosome DIP sites

[0060] Table 9 Primer sequence information table of ATTO-labeled X-DIP site and Amelogenin site

[0061] Example 4 Construction of composite amplification system According to the primers pairs obtained in Example 3 which have good specificity and the amplified fragments are consistent with the expected design, a six-color composite amplification system was constructed. After the establishment of the single site amplification system, the step-by-step superposition method described in Example 3 was used to introduce new sites into the amplification system for testing. At the same time, the primer concentration, annealing temperature, final extension time and temperature, and cycle number of each site in the composite amplification system were carefully adjusted to meet the amplification balance of each site in the composite amplification detection, and the peak height of each site in the typing map reached balance. Through multiple rounds of optimization experiments, the key reaction parameters of the composite amplification system were finally determined, including but not limited to: annealing temperature, amplification cycle number, final extension time, Taq PCR premix amount, total reaction volume, and DNA template amount. It was verified through experiments that when the total reaction volume was 10 μL, stable and balanced detection signals of each amplification site could be obtained. The specific composition and volume ratio of the optimal reaction system are shown in Table 10 below, and the optimal composite PCR amplification reaction program is shown in Table 11 below.

[0062] Table 10 Reaction components and volumes of composite amplification system

[0063] Among them, Taq PCR premix (2x) was purchased from Shengong Bioengineering (Shanghai) Co., Ltd.

[0064] Table 11 Composite PCR amplification reaction program

[0065] Preparation of allelic typing standard This example prepares allelic typing standard (Allelic Ladder) by collecting samples of each X-DIP site showing heterozygous typing in Example 1. The allelic typing standard is constructed using a strategy based on heterozygous samples. The specific preparation steps are as follows: For each X-DIP site, samples showing heterozygous typing are selected for amplification. The unit point PCR amplification reaction program and system are the same as the experimental steps in Example 3. The heterozygous samples (showing double-peak typing maps) of each X-DIP site are obtained by unit point PCR amplification, and the amplification products of the two alleles of the site are obtained. Subsequently, the amplification products of all 50 sites are mixed, and the samples showing heterozygous typing are used to generate a single allelic typing standard for each site. According to the peak height of the single allelic typing standard, the proportion of each single allelic typing standard is adjusted to make the balance of the peak height in the final composite standard better. Finally, a complete allelic typing standard is constructed, and the capillary electrophoresis detection map thereof is shown in Figure 2 , which contains all alleles of 50 X-DIP sites.

[0066] Example 6 Detection of DNA standard and actual human DNA based on the composite amplification system of the present application This example verifies the detection effect of the composite amplification system prepared in Example 4 on DNA samples of different sources. Male DNA standard 9948 (purchased from Wuxi German-American United Biotechnology Co., Ltd.), 1 male human DNA sample and 1 female human DNA sample are used for testing. Amplification is performed according to the optimized composite amplification system and reaction program of Example 4, and capillary electrophoresis and genotyping analysis are performed according to the experimental steps of Example 3.

[0067] The typing map results of male DNA standard 9948, 1 male human DNA sample and 1 female human DNA sample are shown in Figure 3 , Figure 4 and Figure 5 , all sites can be stably detected in all samples, and the peak height balance between sites is good, indicating that the composite amplification system of the present application can meet the detection needs of DNA samples.

[0068] Example 7 Sensitivity of the composite amplification system based on the present application Sensitivity test was performed based on the complex amplification system described in Example 4. DNA standard 9948 was gradient diluted to make the template amount of 1 ng, 500 pg, 250 pg, 125 pg, 62.50 pg and 31.25 pg. Amplification detection was performed according to the amplification system and amplification procedure optimized in Example 4.

[0069] The detection results are shown in Figure 6 . When the template amount is 1 ng, 500 pg, 250 pg, 125 pg and 62.5 pg, the peaks of each detection site can be normally obtained, and the average allele peak height is 3595 RFU, 1778 RFU, 1059 RFU, 566 RFU and 331 RFU, respectively. When the template is as low as 31.25 pg, only 66% of the allele peaks of the detection sites are higher than the analysis threshold (RFU = 100), indicating that the sensitivity of the complex amplification system described in the application is 62.50 pg.

[0070] Example 8 Detection of old degraded samples based on the complex amplification system of the application The application can detect the old degraded biological samples commonly used in forensic practice, such as blood marks, saliva swabs, vaginal secretions and semen stains, etc. Taking a saliva swab sample stored for 11 years by the Southern Medical University Judicial Authentication Center as an example, the detection effect of the complex amplification system of the application in old degraded samples is further illustrated. Two detection methods were used for comparison in this study: the experimental group used the complex amplification system described in Example 4, and the control group used the commercial kit Goldeneye® DNA Identity System 21X. The Chelex-100 method (Walsh et al. 1991) was used to extract genomic DNA from the saliva swab sample, and the amplification was performed according to the optimized complex amplification system and reaction procedure of Example 4. Capillary electrophoresis and genotyping analysis were performed according to the experimental procedures of Example 3.

[0071] The genotyping maps of the saliva swab samples detected by the experimental group and the control group are shown in Figure 7 . The results show that the experimental group can obtain complete genotyping map (peak height range 80-320 RFU), while the control group shows allele loss, and the allele peak height is significantly lower than that of the experimental group (peak height range 20-80 RFU).

[0072] The experimental data show that the complex amplification system described in the application has stronger detection ability in old degraded samples.

[0073] Example 9 Identification of the kinship of half-sister with the same father based on the complex amplification system of the application A group of half-sister relationship identification cases were randomly selected from daily inspection cases of the Identification Center of Southern Medical University as samples. The research objects included a pair of full-sister (D and E) and an individual C who had half-sister relationship with them. The composite amplification system described in Example 4 was used to directly amplify 1 mm of blood sample of the three persons, and the specific operation was performed according to the amplification conditions and capillary electrophoresis detection and analysis method described in Example 3. 2

[0074] The genotyping results are shown in Table 12, which shows that sample C has one same allele as sample D or E at each X-DIP site, which is consistent with the genetic characteristics of half-sister relationship, so the possibility of half-sister relationship between C and D / E cannot be excluded. The experimental results prove that the composite amplification system of the present application can be used for the identification of half-sister relationship of the same father and different mother.

[0075] Table 12 Genotyping results of three individuals with half-sister relationship of the same father and different mother at 50 X-DIP sites

[0076] Note: D: deletion allele; I: insertion allele; D, I: deletion and insertion alleles exist at the same time.

[0077] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.​

Claims

1. A combination of X chromosome DIP sites, characterized in that, The X chromosome DIP site combination comprises 50 X chromosome DIP sites, 2 Y chromosome DIP sites and an Amelogenin identification site; the 50 X chromosome DIP sites are: rs3052378, rs35225800, rs56971671, rs3831664, rs10600022, rs3050111, rs34023916, rs72407549, rs150915048, rs4017281, rs34764424, rs10569568, rs35543200, rs201126330, rs143246211, rs56238119, rs145745343, rs72422496, rs200046032, rs58459443, rs10669433, rs34179015, rs33920981, rs55921940, rs3831660, rs35417817, rs144674109, rs2308239, rs10533508, rs72566727, rs199731653, rs35843349, rs67315811, rs35244442, rs36094418, rs60501705, rs3859989, rs60477615, rs59550361, rs58470327, rs35358831, rs5902767, rs143535638, rs10695276, rs10638624, rs10549472, rs77572119, rs199644583, rs61260787, rs72384910; the two Y chromosome DIP sites are rs759551978 and rs2032678 respectively.

2. Use of a reagent for detecting the X chromosome DIP site combination of claim 1 in parentage identification and individual identification.

3. Use according to claim 2, characterized in that, The reagent comprises a primer pair for amplifying the X chromosome DIP site combination of claim 1; the sequence of the primer pair is shown in SEQ ID NO: 1-100.

4. Use according to claim 3, characterized in that, The concentration of the primer pair is 0.05-1 μM.

5. Use according to claim 4, characterized in that, The 5' end of at least one primer in the primer pair is labeled with a fluorescent dye; the fluorescent dye comprises FAM, HEX, SUM, TAMRA, LYN, ROX, ATTO or PUR.

6. Use according to claim 5, characterized in that, The primer pair is labeled with different fluorescent dyes respectively.

7. Use according to any one of claims 3 to 6, characterized in that, The length of the sequence fragment targeted for amplification by the primer pair is less than 210 bp.

8. Use of the X chromosome DIP site combination according to claim 1 for the preparation of a kit, characterized in that, The kit comprises an allelotyping standard prepared based on the X chromosome DIP site described in claim 1.

9. A method for detecting genetic polymorphism at the DIP locus on the human X chromosome, characterized in that, The method comprises the following steps: using the reagent according to any one of claims 2-7 or the kit according to claim 8 to perform PCR amplification on a sample, and performing genotyping analysis on the PCR amplification product to obtain the genetic polymorphism of the human X chromosome DIP site.

10. The method of claim 9, wherein, The method for performing genotyping analysis on the PCR amplification product comprises capillary electrophoresis or polyacrylamide gel electrophoresis.

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