Compound amplification detection system of X chromosome DIP polymorphic site and application thereof

Abstract

This invention discloses a multiplex amplification detection system for X chromosome DIP polymorphism loci and its applications. The system comprises 50 X chromosome DIP polymorphism loci, one sex identification locus (Amelogenin), and two Y chromosome DIP loci. Of the 50 X chromosome DIP polymorphism loci, 15 can form 6 linkage groups, resulting in higher efficacy for forensic applications. The multiplex amplification detection system of this invention utilizes miniaturized amplicones (70-205 bp) to effectively perform accurate typing analysis of degraded samples in forensic practice. Furthermore, the system is simple, low-cost, and easy to implement, making it significant for individual identification and complex kinship determination in East Asian populations.

Description

Technical Field

[0001] This invention relates to the field of gene detection technology, and in particular to a multiplex amplification detection system for X chromosome DIP polymorphic sites and its application. Background Technology

[0002] The X chromosome, as a crucial sex chromosome in humans, possesses a unique inheritance pattern that holds irreplaceable value in forensic kinship identification. The X chromosome is approximately 155 Mb in length, containing about 1100 genes, accounting for 5% of the coding genes in the human genome. In terms of genetic characteristics, female individuals carry two X chromosomes, one from their father and one from their mother. During meiosis, homologous recombination occurs, and these chromosomes are randomly inherited by offspring. Male individuals, however, carry only one maternal X chromosome, which, except for the pseudoautosomal region, lacks homologous recombination with the Y chromosome and is inherited as a haplotype by their daughters. This sex-linked inheritance characteristic gives the X chromosome a special advantage in complex kinship identification cases, such as grandmother-granddaughter, half-sisters, and cases involving incest: 1. Without considering mutations, grandmothers and granddaughters will always share an identical allele on each X chromosome genetic marker; 2. Half-sisters share the same paternal X chromosome; 3. In paternity tests involving incest, if two disputed fathers are indeed related, they can be considered unrelated individuals for comparison. Studies have shown that in complex kinship identification, such as grandparent-grandchild identification, X chromosome markers often provide crucial supplementary evidence when autosomal markers cannot provide a clear conclusion, highlighting their important supplementary identification value.

[0003] Insertion / deletion polymorphism (DIP) is a special genetic marker characterized by the insertion or deletion of DNA fragments of varying lengths in the human genome. DIP combines the advantages of short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs), making it the most abundant type of DNA polymorphism after SNPs, primarily manifesting as dialexigenicity. Compared to SNPs, DIPs are length polymorphisms, making them suitable for application on the capillary electrophoresis platforms widely used in existing forensic DNA laboratories, offering advantages such as simple typing, ease of operation, and high scalability. Compared to STRs, DIPs have a lower mutation rate, comparable to SNPs, approximately 10-1. -8This makes it more stable. Furthermore, the diallelic structure of DIPs ensures that alleles are known and fixed, and amplicones can be designed into short fragments, suitable for detecting degraded DNA samples. The shortcomings of existing research are mainly as follows: 1. The polymorphism of some X-DIP sites in East Asian populations is poor; 2. The site capacity of existing X-DIP systems is limited (mostly <40 sites); 3. Insufficient mining of X-DIP sites that are closely linked within linkage groups; 4. The amplicon fragments of some systems are too long, which is not conducive to accurate typing detection of degraded samples; 5. The identification efficiency for complex kinship needs further improvement.

[0004] To address the shortcomings of existing X-DIP research, this invention provides a multiplex amplification detection system for X chromosome DIP polymorphic sites and its application. It mainly focuses on screening single X-DIP sites with high polymorphism in East Asian populations, while integrating X-DIP sites linked within linkage groups to further improve the overall polymorphism of the system. Furthermore, it designs short amplicon (approximately 200 bp) to better adapt to the accurate typing of trace degraded samples. Summary of the Invention

[0005] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the objective of the present invention is to provide a multiplex amplification detection system for X chromosome DIP polymorphism sites and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a combination of X chromosome DIP loci, the combination comprising 50 X chromosome DIP loci, 2 Y chromosome DIP loci, and an Amelogenin identification locus, wherein the 50 X chromosome DIP loci are: rs3052378, rs35225800, rs56971671, rs3831664, rs10600022, rs3050111, rs34023916, rs... 72407549, rs150915048, rs4017281, rs34764424, rs10569568, rs35543200, rs201126330, rs1432462 11. rs56238119, rs145745343, rs72422496, rs200046032, rs58459443, rs10669433, rs34179015, rs33 920981, rs55921940, rs3831660, rs35417817, rs144674109, rs2308239, rs10533508, rs72566727, rs 199731653, rs35843349, rs67315811, rs35244442, rs36094418, rs60501705, rs3859989, rs60477615, The two Y chromosome DIP loci are rs759550361, rs58470327, rs35358831, rs5902767, rs143535638, rs10695276, rs10638624, rs10549472, rs77572119, rs199644583, rs61260787, and rs72384910, respectively.

[0008] A second aspect of the invention provides the application of reagents for detecting the above-mentioned combinations of X chromosome DIP sites in paternity testing and individual identification.

[0009] In some embodiments of the present invention, the reagent comprises primer pairs for amplifying the aforementioned molecular marker combination of the X chromosome DIP site.

[0010] In some embodiments of the present invention, the sequences of the primer pairs are as shown in SEQ ID NO:1-100.

[0011] In some embodiments of the present invention, the concentration of the primer pair is 0.05~1 μM.

[0012] In some embodiments of the present invention, the concentration of the primer pair is 0.06~0.9 μM.

[0013] In some embodiments of the present invention, the concentrations of the primer sequences are as follows: the primer pair concentration of SEQ ID NO: 1-2 is 0.2351 μM; the primer pair concentration of SEQ ID NO: 3-4 is 0.1754 μM; the primer pair concentration of SEQ ID NO: 5-6 is 0.2277 μM; the primer pair concentration of SEQ ID NO: 7-8 is 0.1064 μM; the primer pair concentration of SEQ ID NO: 9-10 is 0.1568 μM; the primer pair concentration of SEQ ID NO: 11-12 is 0.2426 μM; the primer pair concentration of SEQ ID NO: 13-14 is 0.1829 μM; the primer pair concentration of SEQ ID NO: 15-16 is 0.2519 μM; the primer pair concentration of SEQ ID NO: 17-18 is 0.1493 μM; the primer pair concentration of SEQ ID NO: 19-20 is 0.1866 μM; SEQ ID NO: 14-15-16 is 0.1493 μM; the primer pair concentration of SEQ ID NO: 19-20 is 0.1866 μM; SEQ ID NO: 14-15-16 is 0.1493 μM; the primer pair concentration of SEQ ID NO: 19-20 is 0.1866 μM; SEQ ID NO: 14-15-16 is 0.1454 ... The primer pair concentrations for SEQ ID NO:21-22 are 0.1735 μM; the primer pair concentrations for SEQ ID NO:23-24 are 0.3676 μM; the primer pair concentrations for SEQ ID NO:25-26 are 0.2202 μM; the primer pair concentrations for SEQ ID NO:27-28 are 0.3396 μM; the primer pair concentrations for SEQ ID NO:29-30 are 0.1978 μM; the primer pair concentrations for SEQ ID NO:31-32 are 0.2855 μM; the primer pair concentrations for SEQ ID NO:33-34 are 0.3004 μM; the primer pair concentrations for SEQ ID NO:35-36 are 0.1922 μM; the primer pair concentrations for SEQ ID NO:37-38 are 0.2146 μM; and the primer pair concentrations for SEQ ID NO:39-40 are 0.3658 μM. The primer pair concentrations for SEQ ID NO:41-42 were 0.3060 μM; the primer pair concentrations for SEQ ID NO:43-44 were 0.2892 μM; the primer pair concentrations for SEQ ID NO:45-46 were 0.2426 μM; the primer pair concentrations for SEQ ID NO:47-48 were 0.2799 μM; the primer pair concentrations for SEQ ID NO:49-50 were 0.2482 μM; the primer pair concentrations for SEQ ID NO:51-52 were 0.2239 μM; the primer pair concentrations for SEQ ID NO:53-54 were 0.2109 μM; the primer pair concentrations for SEQ ID NO:55-56 were 0.2127 μM; the primer pair concentrations for SEQ ID NO:57-58 were 0.4217 μM; and the primer pair concentrations for SEQ ID NO:59-60 were 0.3359 μM. The concentration of primer pair NO:61-62 is 0.3770 μM; the primer pair concentration of SEQ ID NO:63-64 is 0.2202 μM; the primer pair concentration of SEQ ID NO:65-66 is 0.4908 μM; the primer pair concentration of SEQ ID NO:67-68 is 0.3508 μM; the primer pair concentration of SEQ ID NO:69-70 is 0.2874 μM; the primer pair concentration of SEQ ID NO:71-72 is 0.4180 μM; the primer pair concentration of SEQ ID NO:73-74 is 0.2501 μM; the primer pair concentration of SEQ ID NO:75-76 is 0.4684 μM; the primer pair concentration of SEQ ID NO:77-78 is 0.3937 μM; the primer pair concentration of SEQ ID NO:79-80 is 0.3695 μM; SEQ ID The primer pair concentrations for SEQ ID NO: 81-82 were 0.4647 μM; those for SEQ ID NO: 83-84 were 0.3919 μM; those for SEQ ID NO: 85-86 were 0.3134 μM; those for SEQ ID NO: 87-88 were 0.3042 μM; those for SEQ ID NO: 89-90 were 0.5953 μM; those for SEQ ID NO: 91-92 were 0.3266 μM; those for SEQ ID NO: 93-94 were 0.5113 μM; those for SEQ ID NO: 95-96 were 0.5225 μM; those for SEQ ID NO: 97-98 were 0.3191 μM; and those for SEQ ID NO: 99-100 were 0.8724 μM.

[0014] In some embodiments of the present invention, at least one primer in the primer pair is labeled with a fluorescent dye at its 5' end.

[0015] In some embodiments of the present invention, the fluorescent dye includes FAM, HEX, SUM, TAMRA, LYN, ROX, ATTO, or PUR.

[0016] In some embodiments of the present invention, the primer pairs are labeled with different fluorescent dyes.

[0017] In some embodiments of the present invention, the primer pairs shown in SEQ ID NO:1-100, namely the primer pairs shown in 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.

[0018] In some embodiments of the present invention, the primer pairs shown in SEQ ID NO:1-24 are labeled FAM, the primer pairs shown in SEQ ID NO:25-44 are labeled HEX, the primer pairs shown in SEQ ID NO:45-66 are labeled TAMRA, the primer pairs shown in SEQ ID NO:67-84 are labeled ROX, and the primer pairs shown in SEQ ID NO:85-100 are labeled ATTO.

[0019] In some embodiments of the present invention, the primer pair amplifies a sequence fragment with a length of less than 210 bp.

[0020] A third aspect of the present invention is the application of the above-mentioned X chromosome DIP site combination in the preparation of a kit, the kit comprising allelic typing standards prepared based on the above-mentioned X chromosome DIP sites.

[0021] In some embodiments of the present invention, the allele typing standard is an amplification product obtained by amplifying the above-mentioned 50 X chromosome DIP loci individually.

[0022] In some embodiments of the present invention, the amplification product is a heterozygous amplification product.

[0023] In some embodiments of the present invention, the allele typing standard contains all the alleles of the above-mentioned X chromosome DIP locus.

[0024] A fourth aspect of the present invention provides a method for detecting genetic polymorphism at the human X chromosome DIP locus, characterized in that the method comprises the following steps: performing PCR amplification on a sample using the above-mentioned reagents or kits, and performing genotyping analysis on the PCR amplification products to obtain the genetic polymorphism at the human X chromosome DIP locus.

[0025] In some embodiments of the present invention, the amplification system is as follows:

[0026]

[0027] In some embodiments of the present invention, the amplification procedure is as follows:

[0028]

[0029] In some embodiments of the present invention, the method for genotyping analysis of the PCR amplification products includes capillary electrophoresis or polyacrylamide gel electrophoresis.

[0030] The beneficial effects of this invention are:

[0031] This invention provides a multiplex amplification detection system for X-chromosome DIP polymorphism loci and its applications. Of the 50 X-DIP polymorphism loci selected in this invention, 15 loci can form 6 linkage groups. Compared to single X-DIP loci, this system exhibits higher polymorphism, resulting in greater forensic application efficacy. It is more suitable for forensic individual identification and complex kinship determination, and is particularly applicable to individual identification and complex kinship determination in East Asian populations.

[0032] The multiplex amplification detection system targeting X-DIP polymorphic sites described in this invention utilizes miniaturized amplicones (70-205 bp) to effectively perform accurate typing analysis of degraded samples in forensic practice, providing technical support for public security science and technology and scientific evidence for court trials. This multiplex amplification detection system exhibits an efficacy exceeding 0.9999 in paternity testing of duplicates and can play an auxiliary role in complex kinship identification. This invention constructs a simple, low-cost, and easily scalable multiplex amplification detection system suitable for forensic practice, solving the problems of insufficient site capacity and limited polymorphic information in existing X-DIP detection systems. It is of significant importance for assisting forensic individual identification and complex kinship identification. Attached Figure Description

[0033] Figure 1 This diagram shows the fluorescent marker types, amplicon sizes, and site arrangement for the 50 X-DIP sites, the enamel genotype locus (Amelogenin), and the 2 Y-DIP sites of this invention.

[0034] Figure 2 The capillary electrophoresis pattern of the allelic typing standard prepared in this embodiment of the invention.

[0035] Figure 3 This is the typing profile of DNA standard 9948 detected by the multiplex amplification system of this invention.

[0036] Figure 4 This invention provides a typing profile for detecting male human DNA samples using the multiplex amplification system.

[0037] Figure 5 This invention provides a typing profile for detecting human DNA samples from women using the multiplex amplification system.

[0038] Figure 6 This is a graph showing the sensitivity detection results of the multiplex amplification system of this invention.

[0039] Figure 7 This invention provides a genotyping profile of a saliva swab sample preserved for 11 years using the multiplex amplification system. Detailed Implementation

[0040] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the experiments are conventional methods in the art.

[0041] Example 1: Screening of X chromosome DIP polymorphism sites

[0042] Based on X chromosome genomic data from 2504 individuals in the 1000 Genomes Project phase 3 database, a multi-stage screening strategy was employed to identify highly polymorphic X chromosome DIP (X-DIP) loci. Specific screening criteria included:

[0043] 1. Located in an intronic region or intergenic variation region of the X chromosome; 2. Diallelic DIP polymorphism; 3. Insertion / deletion fragment length of 2bp~6bp; 4. Minor allele frequency (MAF) > 0.3 in East Asian populations; 5. Conforms to Hardy-Weinberg equilibrium in East Asian populations; 6. Linked sites must simultaneously satisfy R... 2 >0.2 and D' values ​​>0.5, forming a linkage group; 7. The physical distance between non-linked sites is greater than 0.1 Mb, and the sites are in linkage equilibrium (R0). 2 <0.2); 8. Verification using the NCBI database excluded other polymorphic sites in the flanking sequences 200 bp upstream and downstream of the target site. After the above rigorous screening, the final set of X-DIP candidate sites that meet the requirements is shown in Table 1 below.

[0044] Table 1. Basic information on 50 X-DIP sites

[0045]

[0046] The rs number, reference allele, substitution allele, minor allele frequency (MAF), and physical location are referenced from the dbSNP database (GRCh37). For sex determination, an enamel locus (Amelogenin) and two Y-chromosome DIP (Y-DIP) loci (rs759551978 and rs2032678) are also added for accurate sex identification.

[0047] Example 2: Population genetic analysis of 50 X-DIP loci

[0048] Based on East Asian population data (n=504, 244 males and 260 females) from the 1000 Genomes Project phase 3 dataset, StatsX v2.0 software was used to statistically analyze allele frequencies, linkage haplotype frequencies, and forensic parameters of 50 X-DIP loci. Arlequin software was used for Hardy-Weinberg equilibrium tests, linkage analysis, and sex-specific allele frequency differences. The forensic parameters of each X-DIP locus and linkage group, including the power of discrimination (PD), cumulative power of discrimination (CPD), mean exclusion chance (MEC), and cumulative mean exclusion chance, are shown in Tables 2 and 3 below.

[0049] Table 2. Forensic parameters of 50 X-DIP sites

[0050]

[0051] Note: MEC_Kruger refers to the average exclusion probability calculated using Kruger's method in paternity testing for missing relatives (mother-daughter, grandmother-granddaughter) (Krüger et al., 1968); MEC_Kishida refers to the average exclusion probability calculated using Kishida's method in standard triad paternity testing (including daughters) (Kishida et al., 1997); MEC_Desmarais refers to the average exclusion probability calculated using Desmarais's method in triad paternity testing (including daughters) (Desmarais et al., 1998); MEC_Desmarais_duo refers to the average exclusion probability calculated using Desmarais's method in father-daughter or mother-son paternity testing (Desmarais et al., 1998). (al., 1998); LG1: Chain 1 (rs35244442-rs77572119); LG2: Chain 2 (rs35843349-rs2308239); LG3: Chain 3 (rs35358831-rs60477615-rs72384910); LG4: Chain 4 (rs143535638-rs10695276-rs60501705); LG5: Chain 5 (rs61260787-rs3859989); LG6: Chain 6 (rs34764424-rs10569568-rs10669433).

[0052] Table 3. Cumulative forensic parameters of 50 X-DIP sites

[0053]

[0054] The results showed that there was no statistically significant difference in allele frequencies between male and female populations at all loci, and all loci conformed to Hardy-Weinberg equilibrium after Bonferroni correction.

[0055] Example 3: X-DIP site primer design

[0056] Based on the X-DIP candidate sites screened in Example 1, a systematic primer design process was used to design and evaluate each site.

[0057] 1 Primer Design

[0058] The flanking sequences (200 bp upstream and downstream) of the target DIP site were obtained from the UCSC Human Genome Explorer. Primers were designed using the Primer3 online platform (https: / / primer3.ut.ee / ), following these principles: 1. Primer length was controlled between 20 bp and 28 bp; 2. Amplicon length was between 70 bp and 210 bp; 3. Primer annealing temperature (Tm value) was between 57℃ and 62℃ (optimal Tm value was 60℃), with the Tm value difference between the forward and reverse primers not exceeding 3℃; 4. GC content of the primer sequence was between 30% and 70%, preferably between 40% and 60%; 5. Primer sequences should avoid more than 5 consecutive single base repeats, and primer sequences with more than 3 consecutive C or G at the 3' end should be avoided; 6. In-Silico of the UCSC Human Genome Explorer was used. 7. Use PCR and NCBI's Primer-BLAST function to test the specificity of primer pairs; 8. Use AutoDimer software to assess the risk of primer dimerization, requiring the free energy score of the primers themselves and the interactions between primers to be ≤8.

[0059] 2. Validation and evaluation of primers

[0060] The designed electrophoresis was followed by single-site PCR amplification and then agarose gel electrophoresis to verify the primer specificity and amplification efficiency. The single-site PCR amplification system is shown in Table 4 below.

[0061] Table 4. Reaction components and volume of the unit site amplification system

[0062]

[0063] The Taq PCR premix (2×) was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0064] The specific PCR experimental steps are as follows:

[0065] DNA was extracted using the oral swab genomic DNA extraction kit (TIANGEN BIOTECH) according to the instructions. The DNA was eluted with 40 μL of 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, amplification was performed on an ABI 9700 PCR instrument according to the following reaction procedure:

[0066] (1) Initial denaturation at 95℃ for 2 minutes, repeated once;

[0067] (2) Denaturation at 94℃ for 30 seconds, annealing at 60℃ for 1 minute, extension at 72℃ for 50 seconds, repeat 31 times;

[0068] (3) Extend the final temperature to 60℃ for 60 minutes, and repeat once.

[0069] The amplification products were analyzed using 2% agarose gel electrophoresis. Primer performance was evaluated based on the agarose gel electrophoresis results: the observation of a single, bright, and narrow band indicated good primer specificity and amplification efficiency; weak band signals suggested the need to optimize PCR conditions, including adjusting parameters such as annealing temperature or cycle number; the detection of multiple non-specific bands indicated insufficient primer specificity, requiring primer redesign.

[0070] The 5' ends of the upstream or downstream primers of the primers tested by agarose gel electrophoresis were modified with fluorescence to further verify the primer specificity and whether the amplified fragment length matched the expected design on a capillary electrophoresis platform. The specific experimental steps are as follows:

[0071] (1) Perform PCR according to the above PCR system and reaction procedure;

[0072] (2) The PCR product obtained in (1) was shaken and mixed and then centrifuged briefly. 1 μL of the product was mixed with 0.5 μL of AGCU MarkerSIZ-500 internal standard and 9.5 μL of deionized formamide and added to a 96-well plate. The plate was centrifuged at 2000 rpm for 3 min, denatured at 95℃ for 3 min, and then placed in an ice bath for 3 min.

[0073] (3) Subsequently, the 96-well plate after ice bath (2) was placed on a 31300xL gene analyzer for capillary electrophoresis separation. After electrophoresis, genotyping analysis was performed using GeneMapper® ID-X v1.7 (Applied Biosystems, USA) software. Based on the amplification results of each site, a Bin file was created for each site, where the inserted alleles of the X-DIP site were named I and the deleted alleles were named D. The Bin files of 50 X-DIP sites were then integrated into a Panel file named 50-Plex X-DIP, and this file was imported into the analysis method of the genotyping software GeneMapper to achieve automated genotyping.

[0074] The screening criteria for capillary electrophoresis results were that only a single peak was amplified in male samples, while in female samples, both homozygous single peaks and heterozygous double peaks may appear, with the heterozygous double peaks showing better peak height uniformity. Primer pairs that met the criteria were included in subsequent experiments.

[0075] Based on the above principles and methods, primer sequences for 50 X-DIP sites, Amelogenin, and 2 Y-chromosome DIP (Y-DIP) sites were obtained. Primer sequence information is shown in Tables 5-9 below, and the site arrangement diagram is as follows. Figure 1As shown. All primers were grouped according to the expected amplified fragment length (70-205 bp) (e.g., Figure 1 As shown, five different fluorescent dyes were used to label the X-DIP sites: 6-FAM, HEX, TAMRA, ROX, and ATTO. The first group of X-DIP site primers (SEQ ID NO. 1-2 to SEQ ID NO. 23-24) was 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) was labeled with HEX fluorescent dye; the third group of X-DIP site primers (SEQ ID NO. 45-46 to SEQ ID NO. 65-66) was labeled with TAMRA fluorescent dye; the fourth group of X-DIP site primers (SEQ ID NO. 67-68 to SEQ ID NO. 83-84) was 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) was labeled with ATTO fluorescent dye. Primer sequences (SEQ ID NO. 104-105 and SEQ ID NO. 106-107) for the Y-DIP loci (rs759551978h and rs2032678) were labeled with ROX fluorescent dye, and the 5' end of the upstream primer in all primer pairs was labeled with fluorescent dye. Primer sequences (SEQ ID NO. 100-103) for the enamel locus were labeled with ATTO fluorescent dye.

[0076] Meanwhile, since there are significant differences in the fragment size of the amplification product and the amplification efficiency of the primers at each X-DIP site, new sites are introduced into the amplification system for testing based on the amplification detection results of a single X-DIP site using a stepwise superposition method. That is, primer pairs for new sites are gradually added to the amplification system based on a single site. The specificity of the amplification and whether there is any correlation between primers are evaluated by capillary electrophoresis typing results. The primer pair system for all X-DIP sites is verified and screened. The primer concentration at each site is optimized and adjusted through a series of experiments to gradually improve the amplification balance of the primer combination. Finally, the optimal concentration of each primer pair in the amplification complex detection system is shown in Tables 5-9 below.

[0077] Table 5. Primer sequence information for FAM-labeled X-DIP sites.

[0078]

[0079] Table 6. Primer sequence information for HEX-labeled X-DIP sites.

[0080]

[0081] Table 7. Primer sequence information for HEX-labeled X-DIP sites.

[0082]

[0083] Table 8 Primer sequence information for ROX-labeled X-DIP sites and two Y chromosome DIP sites.

[0084]

[0085] Table 9 Primer sequence information for ATTO-labeled X-DIP sites and Amelogenin sites.

[0086]

[0087] Example 4 Construction of a multiplex amplification system

[0088] A six-color multiplex amplification system was constructed using primer pairs with good specificity and amplified fragments consistent with the expected design obtained in Example 3. After establishing the amplification system for each individual site, new sites were introduced into the amplification system using the stepwise superposition method described in Example 3 for testing. Simultaneously, the primer concentration, annealing temperature, final extension time, temperature, and number of cycles for each site in the multiplex amplification system were meticulously adjusted to ensure balanced amplification at each site during multiplex amplification detection and uniform peak heights in the genotyping spectrum. Through multiple rounds of optimization experiments, the key reaction parameters of the multiplex amplification system were finally determined, including but not limited to: annealing temperature, number of amplification cycles, final extension time, Taq PCR premix volume, total reaction volume, and DNA template amount. Experimental verification showed that when the total reaction volume was 10 μL, stable and balanced detection signals were obtained for each amplification site. The specific composition and volume ratio of this optimal reaction system are shown in Table 10 below, and the optimal multiplex PCR amplification reaction procedure is shown in Table 11 below.

[0089] Table 10. Reaction components and volumes of the multiplex amplification system

[0090]

[0091] The Taq PCR premix (2×) was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0092] Table 11. Procedure for Multiplex PCR Amplification

[0093]

[0094] Example 5 Preparation of allele typing standards

[0095] This embodiment collects samples from each X-DIP locus showing heterozygous genotypes from Example 1 to prepare allelic genotyping standards. A strategy based on heterozygous samples is used to construct the allelic genotyping standards. The specific preparation steps are as follows:

[0096] For each X-DIP locus, samples exhibiting heterozygous genotyping were selected for amplification. The PCR amplification procedure and system for each locus were the same as in Example 3. Heterozygous samples (showing a bimodal genotyping pattern) were obtained from each X-DIP locus through unit-site PCR amplification, yielding amplification products for the two alleles at that locus. Subsequently, the amplification products from all 50 loci were mixed, and a single allele genotyping standard was generated for each locus using samples exhibiting heterozygous genotyping. The proportions of each single allele genotyping standard were adjusted based on the peak height of the single allele genotyping standard to ensure good peak balance in the final composite standard. A complete allele genotyping standard was finally constructed, and its capillary electrophoresis pattern is shown below. Figure 2 As shown, this standard contains all alleles of 50 X-DIP loci.

[0097] Example 6: Detection of DNA Standards and Actual Human DNA Based on the Multiplex Amplification System of the Present Invention

[0098] This embodiment verifies the detection performance of the multiplex amplification system prepared in Example 4 on DNA samples from different sources. Male DNA standard 9948 (purchased from Wuxi Zhongde Meilian Biotechnology Co., Ltd.), one male human DNA sample, and one female human DNA sample were used for testing. Amplification was performed according to the optimized multiplex amplification system and reaction procedure of Example 4, and capillary electrophoresis and genotyping analysis were performed using the same experimental steps as in Example 3.

[0099] The genotyping results of male DNA standard 9948, one male human DNA sample, and one female human DNA sample are as follows: Figure 3 , Figure 4 and Figure 5 As shown, all sites were stably detected in all samples, and the peak heights of each site were well balanced, indicating that the multiplex amplification system of the present invention can meet the detection requirements of DNA samples.

[0100] Example 7 Sensitivity of the multiplex amplification system based on the present invention

[0101] Sensitivity testing was performed using the multiplex amplification system described in Example 4. DNA standard 9948 was serially diluted to obtain template amounts of 1 ng, 500 pg, 250 pg, 125 pg, 62.50 pg, and 31.25 pg. Amplification and detection were performed according to the optimized amplification system and procedure described in Example 4.

[0102] Test results as follows Figure 6 As shown, at template amounts of 1 ng, 500 pg, 250 pg, 125 pg, and 62.5 pg, all detection sites showed normal peaks, with average allele peak heights of 3595 RFU, 1778 RFU, 1059 RFU, 566 RFU, and 331 RFU, respectively. When the template amount was as low as 31.25 pg, only 66% of the detection sites showed allele peak heights higher than the analysis threshold (RFU=100), indicating that the sensitivity of the multiplex amplification system described in this invention is 62.50 pg.

[0103] Example 8: Detection of aged and degraded samples based on the multiplex amplification system of the present invention.

[0104] This invention can detect common, aged, and degraded biological samples used in forensic practice, such as bloodstains, saliva swabs, vaginal secretions, and semen stains. Using a saliva swab sample preserved for 11 years by the Southern Medical University Forensic Science Center as an example, the detection effectiveness of the multiplex amplification system of this invention in aged and degraded samples is further illustrated. This study used two detection methods for genotyping comparison: the experimental group used the multiplex amplification system described in Example 4, while the control group used the commercially available Goldeneye® DNA Identification System 21X kit. Genomic DNA was extracted from the saliva swab samples using the Chelex-100 method (Walsh et al. 1991). Amplification was performed according to the optimized multiplex amplification system and reaction procedure of Example 4. Capillary electrophoresis and genotyping analysis followed the experimental steps in Example 3.

[0105] The genotyping profiles of saliva swab samples from the experimental and control groups are as follows: Figure 7 As shown in the figure. The results showed that the experimental group was able to obtain complete genotyping patterns (peak height range 80-320 RFU), while the control group showed allele loss, and the allele peak heights were significantly lower than those in the experimental group (peak height range 20-80 RFU).

[0106] Experimental data show that the composite amplification system described in this invention has a stronger detection capability in old and degraded samples.

[0107] Example 9: Kinship identification of half-sisters based on the multiplex amplification system of the present invention

[0108] A sample of half-sibling kinship identification cases was randomly selected from the routine cases handled by the Southern Medical University Identification Center. The study subjects included a pair of full sisters (D and E) and an individual C who was a half-sibling to them. The multiplex amplification system described in Example 4 was used to amplify the kinship of the three individuals at 1 mm. 2 The blood sample was directly amplified, and the specific operation was performed according to the amplification conditions and capillary electrophoresis detection and analysis method described in Example 3.

[0109] The genotyping results are shown in Table 12 below, indicating that samples C and D or E share the same allele at each X-DIP locus. This genetic characteristic conforms to the inheritance pattern of half-siblings, therefore the possibility of a half-sibling relationship between C and D / E cannot be ruled out. Experimental results demonstrate that the multiplex amplification system of this invention can be used to identify the kinship of half-siblings.

[0110] Table 12. Typing results of 50 X-DIPs in 3 individuals with a half-sister relationship.

[0111]

[0112] Note: D: Deletion of allele; I: Insertion of allele; D, I: Both deletion and insertion of alleles are present.

[0113] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Application of reagents for detecting combinations of DIP loci on the X chromosome in paternity testing and individual identification; The X chromosome DIP site combination consists of 50 X chromosome DIP sites, 2 Y chromosome DIP sites, and an Amelogenin identification site; The 50 X chromosome DIP loci are: rs3052378, rs35225800, rs56971671, rs3831664, rs10600022, rs3050111, rs34023916, rs72407549, rs150915048, rs4017281, rs34764424, rs10569 568, rs35543200, rs201126330, rs143246211, rs56238119, rs145745343, rs7242249 6. rs200046032, rs58459443, rs10669433, rs34179015, rs33920981, rs55921940, rs 3831660, rs35417817, rs144674109, rs2308239, rs10533508, rs72566727, rs199731 653, rs35843349, rs67315811, rs35244442, rs36094418, rs60501705, rs3859989, rs 60477615, rs59550361, rs58470327, rs35358831, rs5902767, rs143535638, rs10695 276, rs10638624, rs10549472, rs77572119, rs199644583, rs61260787, rs72384910; The two Y chromosome DIP sites are rs759551978 and rs2032678, respectively.

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

3. Use according to claim 2, characterized in that, The concentration of the primer pair is 0.05~1μM.

4. Use according to claim 3, characterized in that, At least one primer in the primer pair is labeled with a fluorescent dye at its 5' end; The fluorescent dyes include FAM, HEX, SUM, TAMRA, LYN, ROX, ATTO, or PUR.

5. The application according to claim 4, characterized in that, The primer pairs are labeled with different fluorescent dyes.

6. The application according to any one of claims 2-5, characterized in that, The primer pair amplifies a sequence fragment with a length of less than 210 bp.

7. The application of X chromosome DIP site combinations in the preparation of kits for paternity testing and individual identification, characterized in that, The kit contains allelic typing standards prepared based on the X chromosome DIP locus; The X chromosome DIP site combination consists of 50 X chromosome DIP sites, 2 Y chromosome DIP sites, and an Amelogenin identification site; The 50 X chromosome DIP loci are: rs3052378, rs35225800, rs56971671, rs3831664, rs10600022, rs3050111, rs34023916, rs72407549, rs150915048, rs4017281, rs34764424, rs10569 568, rs35543200, rs201126330, rs143246211, rs56238119, rs145745343, rs7242249 6. rs200046032, rs58459443, rs10669433, rs34179015, rs33920981, rs55921940, rs 3831660, rs35417817, rs144674109, rs2308239, rs10533508, rs72566727, rs199731 653, rs35843349, rs67315811, rs35244442, rs36094418, rs60501705, rs3859989, rs 60477615, rs59550361, rs58470327, rs35358831, rs5902767, rs143535638, rs10695 276, rs10638624, rs10549472, rs77572119, rs199644583, rs61260787, rs72384910; The two Y chromosome DIP sites are rs759551978 and rs2032678, respectively.

8. A method for detecting genetic polymorphism at the DIP locus on the human X chromosome, characterized in that, The method includes the following steps: performing PCR amplification on the sample using the reagents of any one of claims 1-6 or the kit of claim 7, and performing genotyping analysis on the PCR amplification products to obtain the genetic polymorphism of the human X chromosome DIP site; The method is used for paternity testing and individual identification.

9. The method according to claim 8, characterized in that, The methods for genotyping analysis of the PCR amplification products include capillary electrophoresis or polyacrylamide gel electrophoresis.

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