44 InDel genetic marker system for highly degraded sample typing, detection primer and application of 44 InDel genetic marker system

Abstract

The invention discloses a 44 InDel genetic marker system for highly degraded sample typing, a detection primer and application of the 44 InDel genetic marker system and the detection primer, and belongs to the technical field of medical identification. Through bioinformatics screening and experimental verification, an insertion and deletion (InDel) genetic marker system which is composed of 44 autosomal InDel and a sex identification gene AMEL site and has amplicon length not exceeding 125 bp is constructed, and the genetic marker system is suitable for capillary electrophoresis typing and is specially designed for highly degradable detection materials. The system is simple and convenient to operate, economical and efficient, can remarkably improve the complete typing success rate of the degraded sample, and is convenient to popularize and apply in forensic medicine laboratories. The invention provides a novel technical scheme which is efficient and compatible with a capillary electrophoresis platform for forensic practice.

Description

Technical Field

[0001] This invention relates to the field of forensic identification technology, and in particular to a 44-InDel genetic marker system for typing highly degraded samples, detection primers, and their applications. Background Technology

[0002] Highly degraded specimens are frequently encountered as challenging evidence in forensic practice and a difficult area of ​​forensic research. When biological evidence is exposed to factors such as high temperature, humidity, ultraviolet radiation, microorganisms, strong acids, and strong alkalis, long-chain DNA molecules are broken down into smaller DNA fragments. If the DNA fragmentation in a specimen is extremely severe, even less than 150 bp in length, it can be considered a highly degraded specimen. These specimens are commonly found in hair shafts without follicles, formalin-fixed paraffin-embedded samples, old case evidence, and highly decomposed specimens from large-scale disasters such as air crashes, earthquakes, and tsunamis. Short tandem repeats (STRs) are the most widely used genetic markers in forensic applications; however, due to their relatively large amplicon size and the presence of stutter peaks during genotyping, they are not ideal genetic markers for analyzing highly degraded forensic specimens. Even the miniSTR system, specifically designed for degraded samples, amplifies fragments between 100-330 bp, often yielding no genotyping results when used for identifying highly degraded specimens. The concept of microhaplotype, proposed by KK Kidd in 2014, refers to short sequences containing two or more SNPs and exhibiting haplotype polymorphism. These are considered ideal genetic markers for detecting degraded samples. While microhaplotypes offer advantages such as low mutation rates, short amplicon lengths, and the absence of spurious peaks like shadow peaks during genotyping, their detection relies on massively parallel sequencing (MPS) platforms. The equipment and consumables are expensive, the operation is complex, the detection cycle is long, it requires specialized technicians, and it demands high-quality template DNA. These drawbacks limit its widespread use in forensic DNA laboratories.

[0003] Insertion / deletion polymorphism (InDel) is a DNA polymorphism caused by the insertion or deletion of DNA fragments. Compared with other molecular genetic markers, InDels have the following characteristics: 1) InDel genetic markers are widely present in the human genome, with an average of one InDel every 7.2 kb, second only to SNPs in number; 2) The fragments of a single InDel locus are short, suitable for degrading samples; 3) The mutation rate of InDels is low, approximately 10. -8Furthermore, InDel rarely exhibits repetitive mutations, making it suitable for population genetic polymorphism analysis; 4) InDel fragments range in length from one to several hundred single nucleotides, exhibiting length polymorphism, which can be detected using capillary electrophoresis (CE) platforms, ensuring its application in grassroots forensic laboratories; 5) InDel genetic markers can also be sequenced using massively parallel sequencing platforms, possessing significant potential application and research value. Therefore, InDel combines the advantages of both STRs and SNPs, making it an emerging molecular genetic marker with strong value for forensic science research and applications, suitable for grassroots forensic laboratories. These characteristics make InDel an ideal genetic marker for the identification of highly degraded evidence.

[0004] Currently, due to limitations in the length of the target amplified fragment and the selection of InDel sites, the number of loci that can be accommodated in a single reaction system is limited, and the amount of genetic information that can be covered is also limited. Existing studies are limited by the length of the amplicon or the number of genetic markers within the multiplex amplification system, making it difficult to accurately detect highly degraded samples. Therefore, developing a set of multiplex amplification detection systems with higher identification efficiency suitable for the detection of highly degraded samples is of great significance in actual forensic cases. Summary of the Invention

[0005] The purpose of this invention is to provide a 44 InDel genetic marker system, detection primers, and their applications for typing highly degraded samples, thereby addressing the problems existing in the prior art. This invention provides a system containing 44 InDel genetic markers and... AMEL A comprehensive gene detection system. All amplified fragments are ≤125 bp, suitable for capillary electrophoresis typing, and specifically designed for highly degradable samples. This system is simple to operate, cost-effective, and can significantly improve the success rate of complete typing of degraded samples, making it easy to promote and apply in forensic laboratories.

[0006] To achieve the above objectives, the present invention provides the following solution: The present invention provides a genetic marker system for typing highly degraded samples, characterized in that the genetic marker system consists of the InDel sites located at GRCh37.p13 of the reference genome as shown in Table 1.

[0007] As shown in Table 1, the insertion / deletion sites selected by the genetic marker system are all 2 bases long, and the minimum allele frequency in the Han Chinese population in southern China and the world population is greater than 0.4, thus ensuring that the system can accommodate more sites and achieve better forensic system performance.

[0008] The present invention also provides a primer set for amplifying the genetic marker system, comprising primers with nucleotide sequences as shown in SEQ ID NO.1-88.

[0009] Optionally, the nucleotide sequences of the primer set are shown in Table 2.

[0010] The sequence length amplified by the primers corresponding to each genetic marker is controlled within 125bp, which is suitable for detecting highly degraded biological samples.

[0011] The present invention also provides the application of the primer set described above in the preparation of a reagent kit for forensic identification.

[0012] The present invention also provides a reagent kit for forensic identification, the reagent kit comprising the aforementioned primer set.

[0013] Optionally, the kit may also include a method for amplifying the amelioprotein gene. AMEL Primer pairs for sex identification loci.

[0014] Optionally, the kit may also include a mixture of allele typing standards, DNA standards, and a multiplex amplification reaction mixture.

[0015] The present invention also provides the application of the primer set or the kit described herein in the identification of highly degraded forensic specimens.

[0016] Optionally, the highly degradable specimen is selected from at least one of the following: biological specimens from large-scale catastrophic events, old bones, formalin-fixed paraffin-embedded tissue, and hair without follicles.

[0017] The present invention discloses the following technical effects: This invention enables the identification of 44 insertion / deletion loci on autosomes and one sex determination gene. AMEL The stable, sensitive, specific and efficient multiplex amplification analysis maximizes the number of loci detected and the polymorphism of loci by screening loci, thereby improving the identification efficiency of the multiplex detection system.

[0018] The amplicon length of the 45 loci in this invention is controlled within 125 bp, which enables complete typing of highly degraded biological samples. This largely solves the problem of existing technologies being limited by amplicon length or the number of genetic markers in the multiplex amplification system, and is suitable for the detection and analysis of highly degraded samples.

[0019] The 44 autosomal insertion / deletion loci selected in this invention all involve insertions or deletions of two bases, effectively avoiding the influence of non-template-dependent A-base incompleteness during genotyping and ensuring the maximum number of loci that the system can accommodate. Furthermore, the allele frequencies of these 44 insertion / deletion loci are all greater than 0.4 in both the Han Chinese population in southern China and in 2504 individuals worldwide, effectively ensuring sufficient systemic efficiency of the multiplex amplification system. Linkage analysis of the 44 insertion / deletion genetic markers showed that each marker was in linkage disequilibrium and possessed independent efficiency. Experiments conducted on 180 Han Chinese individuals in Hunan Province demonstrated that the random matching probability of this multiplex detection system is 2.9522 × 10⁻¹⁰. -19 The cumulative non-parent exclusion rate was 0.999854.

[0020] The main technical problem solved by this invention is to realize the use of insertion / deletion genetic markers for forensic individual identification and as an auxiliary tool for paternity testing of highly degraded human biological samples. The composite detection system of this invention comprises a separately packaged mixture of multiplex amplification primers, a mixture of allele typing standards, and DNA standard 9948. The insertion / deletion genetic markers provided by this invention are length polymorphic genetic markers, which can be detected using a capillary electrophoresis platform commonly used in forensic laboratories. They are simple, economical, rapid, and accurate, and easy to promote and apply in forensic practice. All genetic marker amplification products are no longer than 125 bases, providing a simple, economical, rapid, and effective technical means for detecting highly degraded forensic samples in traditional forensic laboratories. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The typing results are for the allelic typing standards of the detection system constructed in this invention; Figure 2 To use the present invention to detect the genotyping results of DNA standard 9948; Figure 3 This invention provides a capillary electrophoresis typing pattern for detecting bloodstains from a Hunan Han Chinese blood sample (sample 344). Figure 4 The image shows the genotyping results detected using the kit from Example 1 of this invention after genomic DNA was incubated at 100°C for 0, 20, 40, 60 and 80 minutes. Figure 5The image shows the genotyping results of the SureID panglobalSTR kit after the genomic DNA was incubated at 100°C for 0, 20, 40, 60 and 80 min. Figure 6 The image shows the genotyping results of the genomic DNA after it has been in a water bath at 100°C for 60 minutes, detected using the kit from Example 1 of this invention. Figure 7 This is a comparison chart of the allele detection rates when detecting artificially degraded DNA using the kit from Example 1 of this invention and the SureID panglobal STR kit. Figure 8 A graph showing the typing results of blood DNA detected using the kit of Example 1 of this invention; Figure 9 A diagram showing the typing results of DNA from paraffin-embedded tissue detected using the kit from Example 1 of this invention; Figure 10 Image showing the genotyping results of DNA detection in paraffin-embedded tissue using the SureID panglobal STR kit; Figure 11 To detect the genotyping pattern of the daughter using the kit of Example 1 of the present invention; Figure 12 To detect the maternal typing using the kit of Example 1 of the present invention; Figure 13 To detect the genotyping profile of the tested father using the kit of Example 1 of the present invention. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] This invention establishes a detection system for highly degraded samples with multiplex insertion and deletion genetic markers by screening ideal insertion and deletion genetic markers from the NCBI database, designing multiplex amplification primers with amplification fragments of no more than 125 bases, using multiplex amplification technology to obtain the alleles of these genetic markers in the sample, obtaining allele typing results by capillary electrophoresis, and finally establishing a system for detecting highly degraded samples with multiplex insertion and deletion genetic markers.

[0029] The screening criteria for multiple insertion / deletion genetic markers in this invention are as follows: 1) The minimum allele frequency (MAF) of the insertion / deletion genetic marker was >0.4 in the Han Chinese population in southern China and in 2504 individuals worldwide; 2) Located in introns or non-coding regions, and unrelated to gene expression; 3) The length of the inserted or deleted nucleotide sequence is 2 bp; 4) Candidate loci must be in linkage equilibrium, with physical distances between loci located on different chromosomes or on the same chromosome exceeding 10 Mb; 5) The size of the PCR product should not exceed 125 bp; 6) Nucleotide sequences with no special structure at the flanking positions of the locus or other genetic markers that affect locus typing detection.

[0030] Based on the established criteria, this invention screened out 44 ideal insertion / deletion genetic markers and established a composite detection system. Information on the 44 insertion / deletion genetic markers is shown in Table 1 (reference genome is GRCh37.p13), and the nucleotide sequences of each primer in the corresponding amplification primer mixture are shown in Table 2.

[0031] Table 1. Insertion / deletion sites included in the composite detection system Table 2. Nucleotide sequences of amplification primers Note: Locus 45 AMEL is the ammonia protein gene. AMEL For sex identification, there is a single nucleotide polymorphism in the upstream primer binding region of this site. Therefore, two upstream primers were designed to ensure amplification efficiency.

[0032] Based on the above results, this invention constructs a multiple insertion and deletion genetic marker detection kit for typing highly degraded samples, which detects the above 45 multiple insertion and deletion genetic markers.

[0033] The kit of this invention specifically comprises the following components: a mixture of isolated and packaged multiplex amplification primers (Table 2, including primers for 44 multiple insertion / deletion genetic markers and one primer for a sex identification gene), a mixture of allele typing standards, DNA standards, and a multiplex amplification reaction mixture; The allele typing standard mixture consists of allele standards from 45 loci (including 44 multiple insertion / deletion genetic markers and one sex identification gene). Figure 1 It consists of 90 fluorescently labeled DNA fragments (corresponding to all 90 known alleles at 45 loci, see Table 3 for details); The DNA standard is standard DNA 9948; The multiplex amplification reaction mixture contains commonly used components such as PCR buffer solution, MgCl2, dNTPs, and DNA polymerase; in a specific embodiment of the present invention, the amplification reaction mixture used is QIAGEN Multiplex PCR mix manufactured by QIAGEN GmbH, Germany.

[0034] Internal molecular weight standard: Size 500, Ningbo Haier Gene Technology Co., Ltd.

[0035] The working principle of this kit is as follows: First, genomic DNA is extracted from highly degraded samples. The DNA template is then mixed with a primer mixture containing 44 insertion / deletion genetic markers and one sex-identifying gene, along with an amplification reaction mixture. PCR amplification is then performed in a single tube, simultaneously obtaining allele amplification products of the 44 multiple insertion / deletion genetic markers and the sex-identifying gene. Finally, capillary electrophoresis is performed, and a mixture of allele typing standards is used as a control to obtain the typing results for the highly degraded samples. In addition, DNA standard 9948 is used as a positive control and amplified simultaneously with the samples. The accuracy of the typing results can be assessed based on the standard DNA. Figure 2 ), to determine whether the test results are reliable.

[0036] Currently, the detection methods for highly degradable samples are not mature enough, and there is a lack of methods that can be widely adopted in most forensic laboratories. This invention's composite detection system aims to establish a simple, economical, convenient, effective, mature, and widely applicable new method for detecting highly degradable samples, for application in forensic practice. In this invention, as the number of primers in the composite amplification system increases, avoiding interference between primers from different loci is a challenge in constructing this highly degradable sample composite detection system. This invention considers the following factors: 1) The length ranges of amplified fragments between multiple insertion / deletion genetic markers with the same fluorescent color do not overlap, so as to distinguish the allele peaks of different genetic markers; 2) The length of the amplified fragment is between 70-125 bp, which is suitable for the detection of highly degraded samples; 3) The primer annealing temperatures are similar; 4) There are no obvious mismatches, hairpin structures, or dimer structures in the primers themselves, between primers, or between primers and template.

[0037] Table 2 shows the primer sequences for the multiplex amplification of all 44 insertion / deletion genetic markers and one sex identification gene.

[0038] Allele typing standard mixtures facilitate accurate and rapid analysis of sample genotypes. This invention names alleles for multiple insertion / deletion genetic markers, with the number "0" indicating a "deleted" allele and "2" indicating an "inserted" allele. Allele information is shown in Table 3.

[0039] Table 3. Nucleotide sequences of allele standards in the allele standard mixture. The kit described in this invention can be used to analyze highly degraded biological samples. The specific analysis method is as follows: (1) Extract DNA from the highly degraded sample to be tested as an amplification template; (2) The DNA extracted in step (1) was subjected to single-tube multiplex amplification using the amplification primer mixture and amplification reaction mixture described above. The multiplex amplification system was prepared as follows: 5 μL total reaction volume, containing 2.5 μL QIAGEN Multiplex PCR mix, 0.8 μL multiplex amplification primer mixture, 0.5 μL DNA template, and 1.2 μL deionized water. The cycling parameters for the multiplex amplification PCR reaction were: 95℃, 5 min; 95℃, 30 s, 59℃, 90 s, 72℃, 30 s, 30 cycles; then 68℃, 80 min; stored at 4℃. Note: Highly degraded DNA exhibits a random effect during amplification; 3-5 parallel amplifications are required for the PCR reaction.

[0040] (3) The amplification products and allele typing standard mixtures were mixed with molecular weight internal standard and Hi-Di formamide respectively and then subjected to capillary electrophoresis. The genotype of the sample was obtained based on the electrophoresis results.

[0041] The following specific examples further illustrate this point. The main reagents and instruments used in this invention are as follows: 1) Automated Laser Fluorescence Capillary Electrophoresis 3130 Genetic Analyzer, ABI Corporation; 2) T100 TM PCR amplification instrument, BIO-RAD Corporation; 3) High-speed centrifuges from Thermo Electron LED GmbH and ThermoFisher. 4) NanoDrop one ultra-micro volume spectrophotometer, Thermo Scientific; 5) Pipette, Eppendorf; 6) Hi-Di formamide, ABI Pharmaceuticals; 7) Internal molecular weight standard (Size 500), Ningbo Haier Gene Technology Co., Ltd.; 8) Universal column-based DNA extraction kit, Guangzhou Meiji Biotechnology Co., Ltd.; 9) DNA extraction kit for paraffin tissue sections, Guangzhou Meiji Biotechnology Co., Ltd.; To further understand the invention and its advantages, the advantages of the invention will be described in detail below with reference to six specific embodiments.

[0042] Example 1: Preparation of the reagent kit The Human Autosomal 44 Insertion / Deletion Loci Combined Sex Identification Gene Complex Detection Kit for Highly Degraded Sample Typing includes the following reagents packaged separately: a) Multiplex amplification primer mixture. As shown in Table 2, all amplification primers were synthesized by Sangon Biotech. The synthesized amplification primers were prepared with ultrapure water to a concentration of 100 pmol / μL, and then mixed according to the proportions shown in Table 4 to prepare the multiplex amplification primer mixture.

[0043] b) Multiplex PCR mix. In this example, QIAGEN MultiplexPCR mix manufactured by QIAGEN AG, Germany was used.

[0044] c) Allelic typing standard mixture: composed of 90 fluorescently labeled DNA fragments, namely the amplification products of mixed pool DNA composed of the primer pairs in Table 2 from 100 Han Chinese individuals from southern China, with four colors (blue, green, black, and red) fluorescence, namely FAM, HEX, TAMRA, and ROX.

[0045] The reagent kit is prepared by packaging the above reagents according to their respective standard requirements and then using them for subsequent experiments.

[0046] Table 4 Concentration of Primers for Multiplex Amplification Example 2: Detection of a Hunan Han Chinese sample using the kit of the present invention. A bloodstain sample from a Han Chinese population in Hunan Province was tested using the aforementioned human autosomal 44 insertion / deletion loci combined with a sex determination gene detection kit for typing highly degraded samples. The specific testing procedure is as follows: a. The DNA extracted using a universal column-based DNA extraction kit was quantified using a NanoDrop one ultra-micro spectrophotometer and then diluted to 2 ng / µL to serve as a template for multiplex amplification. b. Using the DNA template from step a, the multiplex amplification primer mixture, and the multiplex amplification reaction mixture, perform multiplex PCR amplification of the sample in the amplification system shown in Table 5.

[0047] Table 5 Amplification System The cycling parameters for the multiplex amplification PCR reaction were: 95℃, 5 min; 95℃, 30 s, 59℃, 90 s, 72℃, 30 s, 30 cycles; then 68℃, 80 min; and stored at 4℃.

[0048] c. Capillary electrophoresis 1 µL of each amplification product and allele genotyping standard was added to 9 µL of Hi-Di formamide and 0.2 µL of Size 500 molecular weight internal standard, respectively, and mixed thoroughly. Electrophoresis was performed using an ABI Genetic Analyzer 3130 (USA). Electrophoresis conditions: 15 kV, 36 cm capillary tube, POP4 gel, 30 min. Genotyping data were analyzed using GeneMapper IDX v1.4 software (Applied Biosystems, Foster City, CA, USA).

[0049] Using the kit from Example 1, the above-described detection process was followed to perform genotyping on unrelated individuals of the Southern Han ethnicity from Hunan Province, obtaining complete alleles. Figure 3 This is the genotyping result of a Han Chinese sample from Hunan. The genotyping of all 44 insertion / deletion genetic markers and one sex identification gene showed no interfering peaks, and the alleles of the 45 loci in this sample could be clearly identified.

[0050] Example 3: Detection of artificially degraded DNA using the kit of the present invention The artificial degradation model was created using a 2 ng / µL DNA sample (a random sample from the Hunan Han population, distinct from Example 2) in a 100°C water bath for specific time periods (0 min, 20 min, 40 min, 60 min, and 80 min). Genotyping of the artificial degradation products was performed using the kit from Example 1 and the SureID panglobal STR kit. Results are shown below. Figures 4-7 .

[0051] Figure 4 The kit from Example 1 was used to detect DNA typing results at different levels of degradation. Figure 5 The results of the typing of DNA from the SureID panglobal STR kit, commonly used in forensic medicine, show that the SureID panglobal STR kit could not obtain complete typing after detecting DNA boiled for 40 minutes, while the kit in Example 1 could still obtain complete typing after detecting DNA boiled for 60 minutes. The SureID panglobal STR kit had only a 24% allele detection rate after detecting DNA boiled for 60 minutes. Figure 6 The kit from Example 1 was used to detect the genotyping results of DNA degradation after boiling in a water bath for 60 min. All allele peaks could be observed. Figure 7The graph shows a comparison of the allele detection rate of the kit in Example 1 and the SureID panglobal STR kit when detecting artificially degraded DNA. It can be seen that the kit in Example 1 of the present invention is significantly better than the SureID panglobal STR kit when detecting highly degraded DNA, and can be well applied to the detection of highly degraded samples.

[0052] Example 4: Detection of DNA from paraffin-embedded tissue using the kit of the present invention. DNA was extracted from paraffin-embedded tissue (heart tissue sample provided by Xiangya Forensic Science Center, Hunan Province) using a paraffin tissue section DNA extraction kit. Blood DNA was extracted from the same individual as the paraffin-embedded tissue using a universal column-based DNA extraction kit. Genotyping of the paraffin-embedded tissue DNA and blood DNA was performed using the composite detection system of this invention and the SureID panglobal STR kit. The results are shown below. Figures 8-10 .

[0053] Figure 8 The results of blood DNA typing were obtained using the kit in Example 1 of this invention. Figure 9 The results of DNA typing of paraffin-embedded tissues using the composite detection system of this invention were compared. Both methods showed all allele peaks and consistent typing results, indicating that the kit can accurately detect DNA typing of paraffin-embedded tissues. Figure 10 The results of DNA typing of paraffin-embedded tissue using the SureID panglobal STR kit show that only some short amplicon sites were genotyped. This demonstrates that the kit in Example 1 is significantly superior to the SureID panglobal STR kit in detecting highly degraded DNA in practice.

[0054] from Figures 8-10 As can be seen, the DNA in paraffin-embedded tissue is highly degraded. The SureID panglobal STR kit can only detect a few alleles in paraffin-embedded tissue, while the kit of Example 1 of this invention can still detect all 45 alleles. This shows that the kit of this invention has a significantly better ability to obtain complete typing from highly degraded samples in practical applications than conventional STR kits, providing a new method for typing highly degraded samples.

[0055] Example 5: Calculation of population genetic parameters of the kit of the present invention Using the kit of this invention, genotyping was performed on 180 unrelated individuals of Southern Han ethnicity from Hunan Province according to the detection process in Example 2, and complete alleles were obtained.

[0056] Using STRAF and Arlequin v3.5 software, the allele frequencies (Table 6) and forensic parameters (Table 6) of the above 44 insertion / deletion genetic markers were calculated, including the matching probability (MP), polymorphism information content (PIC), exclusion probability (PE), typical paternity index (TPI), expected heterozygosity (He), observed heterozygosity (Ho), and Hardy-Weinberg equilibrium p-value (HWE-p).

[0057] Table 6. Allele frequencies of 44 insertion / deletion loci in 180 unrelated Han Chinese individuals from southern China. Table 7. Forensic parameters of 44 insertion / deletion loci. As shown in Table 6, all 44 insertion / deletion (InDel) genetic markers are evenly distributed in the Han Chinese population in southern China and have a high degree of heterozygosity, which is very valuable for ensuring the efficacy of the reagent kit system.

[0058] As shown in Table 7, the MP values ​​of the 44 loci ranged from 0.3446 (R10) to 0.4271 (R6), indicating that the cumulative random matching probability of the 44 insertion / deletion genetic markers was as low as 10. -19 The PIC values ​​for all loci ranged from 0.3581 (Y2) to 0.375 (R6), indicating a high polymorphism content as dialleles. The Ho and He values ​​for all loci were close and between 0.4 and 0.6, suggesting high heterozygosity at these loci and that the observed data were consistent with the expected genetic equilibrium.

[0059] Meanwhile, the probability of non-paternity exclusion (PE) is a direct indicator of the efficacy of paternity testing. In Table 7, the PE values ​​for the 26 loci ranged from 0.1173 (R10) to 0.2714 (R6). The 44 insertion / deletion loci collectively contributed to a cumulative non-paternity exclusion rate as high as 0.999854. The typical paternity index (TPI) reflects the ability of a locus to support a paternal relationship when the mother is confirmed to be heterozygous. Nearly half of the 44 insertion / deletion loci had TPIs greater than 1, with loci such as R11 (1.1392) and R6 (1.2) providing strong supporting evidence.

[0060] Table 7 also shows the good genetic quality of 44 markers. Hardy-Weinberg equilibrium (HWE-p value): R10 (p=0.0162) and R6 (p=0.0363) deviated from equilibrium at the 0.05 level, but both were greater than the Bonferroni-corrected p value of 0.0011 (0.05 / 44). The p values ​​of the remaining 42 loci (88.5%) were all greater than 0.05, indicating that these genetic markers were in genetic equilibrium in this Hunan Han population and were not significantly disturbed by factors such as obvious population substructure, selection, or mutation, making the data reliable.

[0061] Example 6: Application of Triplets in Paternity Testing The client (daughter, biological mother) requests a paternity test with the father being tested. The client provides blood cards (FTA cards) corresponding to the three individuals as biological samples to be tested.

[0062] The method of Example 2 was used to perform genotyping with the kit of Example 1 to obtain complete allele typing. Figures 11-13 ).

[0063] According to the "GA / T 965-2011 Standard for Forensic DNA Paternity Testing", based on the allele frequencies obtained from the population survey of 180 unrelated Han Chinese individuals in southern China in Example 5 (see Table 6), the paternity index (PI) and cumulative paternity index (CPI) of the triplet at each insertion / deletion locus were calculated.

[0064] Table 8 shows the typing results of the tested father, biological mother and daughter in this triplete. The results show that all multiple insertion and deletion loci in this triplete conform to Mendelian inheritance. The cumulative paternity index (CPI) of this triplete was calculated to be 13163.8052, which proves that the kit of Example 1 can be successfully applied to paternity testing of highly degraded samples or as an auxiliary tool for paternity testing.

[0065] Table 8. Paternity index of the triad at 44 insertion / deletion loci. The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A genetic marker system for typing highly degraded samples, characterized in that, The genetic marker system consists of the InDel sites located at GRCh37.p13 of the reference genome, as shown in the table below: 。 2. A primer set for amplifying the genetic marker system of claim 1, characterized in that, This includes primers with nucleotide sequences as shown in SEQ ID NO.1-88.

3. The use of the primer set according to claim 2 in the preparation of a reagent kit for forensic identification.

4. A reagent kit for forensic identification, characterized in that, The kit includes the primer set as described in claim 2.

5. The reagent kit according to claim 4, characterized in that, The kit also includes a method for amplifying the amber gene. AMEL Primer pairs for sex identification loci.

6. The reagent kit according to claim 4, characterized in that, The kit also includes a mixture of allele typing standards, DNA standards, and a multiplex amplification reaction mixture.

7. The application of the primer set according to claim 2 or the kit according to any one of claims 4-6 in the identification of highly degradable forensic specimens.

8. The application according to claim 7, characterized in that, The highly degradable specimens are selected from at least one of the following: biological specimens from large-scale catastrophic events, old bones, formalin-fixed paraffin-embedded tissues, and hair without follicles.

9. The application of the primer set according to claim 2 or the kit according to any one of claims 4-6 in individual identification or kinship identification.

10. The application according to claim 9, characterized in that, The DNA of the biological sample to be tested is amplified by multiplex PCR using the primer set described in claim 2 or the kit described in any one of claims 4-6 to obtain amplification products; the amplification products are detected by capillary electrophoresis; based on the capillary electrophoresis results, the genotype of the biological sample to be tested at the InDel site described in claim 1 is analyzed; based on the genotype analysis results, the biological sample to be tested is used for individual identification or kinship determination.

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