A 26-multi-inde genetic marker system for highly degradable sample typing, detection primer and application thereof

Abstract

The application discloses a 26 Multi-InDel genetic marker system for highly degraded sample typing, detection primers and application thereof, and belongs to the technical field of forensic identification. The application solves the problem that the detection of degraded samples and high system efficiency are difficult to be considered by constructing a 26 Multi-InDel genetic marker system with all amplicon lengths being not more than 125 bp through bioinformatics screening and experimental verification. ‑17 The random matching probability of the system in a Hunan Han population reaches 2.25*10 The cumulative non-paternity exclusion rate is 0.999925, complete typing of highly degraded DNA can be realized, and triad paternity identification can be completed, and the application provides a new technical scheme of high efficiency and compatible 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 26-multi-InDel genetic marker system for typing highly degradable samples, detection primers, and their applications. Background Technology

[0002] With the increasing demand for forensic evidence identification in scenarios such as large-scale catastrophic events and cold cases, the detection of highly degraded DNA samples has become a key challenge in forensic practice. The DNA in these samples is often severely fragmented, with lengths mostly below 150 bp, making it difficult to obtain complete information using conventional short tandem repeat (STR) typing techniques. While existing STR typing techniques based on capillary electrophoresis (CE) are the gold standard for forensic individual identification, their amplicon lengths are typically above 100 bp, limiting their coverage of highly degraded samples. Even the miniSTR system, designed specifically for degraded samples, generally amplifies fragments between 100-330 bp, only suitable for mildly to moderately degraded samples, and cannot meet the need for effective typing of highly degraded samples with even shorter DNA fragments.

[0003] To address this challenge, researchers have turned to developing detection systems based on single nucleotide polymorphisms (SNPs) and insertion / deletion polymorphisms (InDels). These markers, due to their short core sequences and low mutation rates, are more suitable for highly degraded samples. Microhaplotype markers, in particular, exhibit higher polymorphism, but their genotyping relies on next-generation sequencing platforms, limiting their application in traditional forensic laboratories. Meanwhile, multi-InDels, as length polymorphism markers, can be detected via capillary electrophoresis, better suited to the technical capabilities of existing laboratories, and have become one of the ideal choices for genotyping highly degraded samples. Currently, some studies have developed multiplex amplification systems based on Multi-InDels and applied for related patents, such as a detection kit based on 18 multiplex insertion / deletion genetic markers. However, these systems mostly use five-color fluorescent labeling, which, limited by the types of fluorophores and the length range of amplified fragments, can only accommodate a limited number of loci, resulting in insufficient system efficiency. This makes it difficult to simultaneously meet the stringent requirements of individual identification ability and non-paternity exclusion rates in applications such as highly degraded sample detection and forensic paternity testing.

[0004] In general, existing technologies have two main limitations: firstly, conventional STR and miniSTR kits cannot achieve comprehensive typing of highly degraded DNA due to the excessively long amplified fragments; secondly, the reported Multi-InDel detection systems incorporate a limited number of genetic markers, resulting in low system efficiency and failing to meet the detection needs of degraded samples while simultaneously meeting the requirements of forensic identification regarding random matching probability, cumulative non-paternity exclusion rate, and other indicators. Therefore, developing a genetic marker system and detection method capable of accurately typing highly degraded samples on a capillary electrophoresis platform and possessing sufficient forensic system efficiency has become a pressing technical challenge in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a Multi-InDel genetic marker system, detection primers, and their applications for genotyping highly degraded samples, thereby addressing the problems existing in the prior art. This invention successfully constructed a Multi-InDel marker system with 26 amplicon markers, all ≤125bp, solving the challenge of simultaneously achieving detection of degraded samples and high system efficiency. The system achieved a random matching probability of 2.25 × 10⁻⁶ in the Han Chinese population of Hunan Province. -17 The cumulative non-paternity exclusion rate is 0.999925. It can completely genotype highly degraded DNA and complete triplet paternity testing, providing a new technical solution for forensic practice that is efficient and compatible with capillary electrophoresis platforms.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a genetic marker system for typing highly degraded samples, the genetic marker system consisting of Multi-InDel sites located at GRCh37.p13 of the reference genome as shown in the table below:

[0008] .

[0009] The present invention also provides a primer set for amplifying the genetic marker system, which consists of primers with nucleotide sequences as shown in SEQ ID NO.1-30 and SEQ ID NO.33-56, respectively.

[0010] The present invention also provides the application of the primer set described above in the preparation of a reagent kit for forensic identification.

[0011] The present invention also provides a reagent kit for forensic identification, the reagent kit comprising the aforementioned primer set.

[0012] Optionally, the kit may further include primer pairs for amplifying the AMEL sex identification site of the amelioprotein gene, the nucleotide sequences of which are shown in SEQ ID NO.31-32.

[0013] Optionally, the kit may also include a mixture of allele typing standards, DNA standards, and a multiplex amplification reaction mixture.

[0014] The present invention also provides the application of the primer set or the kit described herein in the identification of highly degraded forensic specimens.

[0015] 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.

[0016] The present invention also provides the application of the primer set or the kit described herein in individual identification or kinship identification.

[0017] Furthermore, the DNA of the biological sample to be tested is amplified by multiplex PCR using the primer set or the kit 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 Multi-InDel site is analyzed; based on the genotype analysis results, the biological sample to be tested is used for individual identification or kinship determination.

[0018] The present invention discloses the following technical effects:

[0019] This invention successfully resolves the core contradiction in existing technologies where it is difficult to simultaneously achieve high detection rates for highly degraded samples and high efficiency in forensic systems. Through bioinformatics screening and experimental verification, a multi-InDel genetic marker system consisting of 26 amplicon lengths not exceeding 125 bp was constructed. This system not only has a short core sequence that conforms to the fragmentation characteristics of highly degraded DNA, but also exhibits higher polymorphism than a single InDel due to its "multiple" nature. Therefore, while ensuring a high detection rate for degraded samples, it achieves excellent individual identification capabilities (random matching probability of 2.25 × 10⁻⁶). -17 ) and the efficacy of paternity testing (cumulative non-father exclusion rate of 0.999925).

[0020] This invention is the first to efficiently integrate 26 Multi-InDel markers into a single six-color fluorescent multiplex amplification system, overcoming the limitations of previous systems with limited genetic marker numbers and insufficient system efficiency. The kit is compatible with capillary electrophoresis platforms commonly used in forensic laboratories, offering ease of operation and cost-effectiveness. Practical application verification shows that it can achieve complete typing of difficult samples such as artificially degraded DNA and paraffin-embedded tissue, and has been successfully applied to triplet paternity testing, accumulating paternity indices to the millions, demonstrating a significant ability to exclude non-biological fathers. Therefore, this invention provides the forensic field with a novel method for detecting highly degraded samples that combines high sensitivity, high discriminative power, and ease of application. 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;

[0023] Figure 2 To use the present invention to detect the genotyping results of DNA standard 9948;

[0024] Figure 3 This invention provides a kit for detecting the capillary electrophoretic typing pattern of 199-C bloodstain samples from Han Chinese populations in Hunan Province.

[0025] Figures 1-3 In the figure, the horizontal axis represents the length of the amplified fragment, the allele peaks are named with respect to the genetic markers, the Arabic numerals below the allele peaks are named with respect to the alleles of the corresponding genetic markers, and the vertical axis represents the fluorescence signal intensity.

[0026] Figure 4 The kit used in Example 1 was used to detect the typing results of DNA with different degrees of degradation;

[0027] Figure 5 The SureID panglobal STR kit was used to detect the typing results of DNA with different degrees of degradation.

[0028] Figure 6 The kit used in Example 1 was used to detect the DNA degradation typing results after boiling in a water bath for 60 min.

[0029] Figure 7This is a comparison chart of the allele detection rates when detecting artificially degraded DNA using the kit from Example 1 and the SureID panglobal STR kit.

[0030] Figure 8 The results of blood DNA typing were obtained using the kit from Example 1.

[0031] Figure 9 The kit used in Example 1 was used to detect the DNA typing results of paraffin-embedded tissues;

[0032] Figure 10 The results of DNA typing in paraffin-embedded tissue using the SureID panglobal STR kit;

[0033] Figure 11 This is a typing diagram of the test subject based on the kit from Example 1;

[0034] Figure 12 The genotyping diagram of the daughter using the kit from Example 1 is shown.

[0035] Figure 13 This is a typing diagram of the mother using the kit from Example 1. Detailed Implementation

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] This invention screens ideal multiple insertion and deletion genetic markers from the NCBI database, designs multiplex amplification primers with amplification fragments of no more than 125 bases, uses multiplex amplification technology to obtain the alleles of these genetic markers in the sample, and obtains the allele typing results by capillary electrophoresis technology, and finally establishes a detection system for highly degraded samples with multiple insertion and deletion genetic markers.

[0042] The screening criteria for multiple insertion / deletion genetic markers in this invention are as follows:

[0043] 1) The minimum allele frequency (MAF) of a single InDel locus contained in a multiple insertion / deletion genetic marker is >0.1;

[0044] 2) In the non-encoded area;

[0045] 3) The maximum physical distance between two InDel loci within a multiple insertion / deletion genetic marker is less than 70 base pairs;

[0046] 4) The difference between the smallest and largest allele segments within the same genetic marker does not exceed 12 bases;

[0047] 5) The size of the PCR product should not exceed 125 bp;

[0048] 6) The physical distance between multiple insertion / deletion genetic markers on the same chromosome is greater than 5 Mb;

[0049] 7) The allele frequency difference between the InDel loci is greater than 0.08.

[0050] Based on the established criteria, this invention screened out 19 ideal multiple insertion / deletion genetic markers and combined them with 7 short amplicon high-polymorphism multiple insertion / deletion genetic markers reported in previous studies to establish a composite genetic marker system. Information on the 26 multiple insertion / deletion genetic markers involved 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.

[0051] Table 1. Insertion / deletion sites included in multiple insertion / deletion genetic markers.

[0052]

[0053] The "*" symbol marks multiple insertion / deletion sites reported in previous studies.

[0054] Table 2. Nucleotide sequences of amplification primers

[0055]

[0056] The "**" symbol marks the multiple insertion / deletion sites for sex identification and the corresponding primer numbers.

[0057] Based on the above results, this invention constructs a six-color fluorescent multiple insertion / deletion genetic marker detection kit for typing highly degraded samples, targeting the above 26 multiple insertion / deletion genetic markers.

[0058] The kit of this invention specifically comprises the following components: a mixture of isolated and packaged multiplex amplification primers (Table 2, including primers for 26 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;

[0059] The allele typing standard mixture consists of allele standards from 27 loci (including 26 multiple insertion / deletion genetic markers and one sex identification gene). Figure 1 It consists of 80 fluorescently labeled DNA fragments (26 pairs of primers amplify 26 multi-indel sites, each site yields three amplicons, plus two sex amplicons amplified by primers for sex identification sites, see Table 8 below).

[0060] The DNA standard is standard DNA 9948;

[0061] 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 a PCR mix produced by Qilin Intelligent Cell Biotechnology (Suzhou) Co., Ltd.

[0062] Internal molecular weight standard: SD550, Suzhou Qilin Intelligent Cell Biotechnology Co., Ltd.

[0063] The working principle of this kit is as follows: First, genomic DNA is extracted from highly degraded samples. The DNA template is mixed with primers containing 26 multiple insertion / deletion genetic markers (as listed in Table 1) plus a sex-identifying gene, along with the amplification reaction mixture. PCR amplification is then performed in a single tube, simultaneously obtaining allele amplification products of the 26 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 typing results can be determined based on the standard DNA... Figure 2 To determine whether the test results are accurate and reliable, we need to assess their accuracy.

[0064] Currently, the detection methods available for highly degradable samples are not mature enough. The kit of this invention aims to establish a simple, economical, convenient, effective, mature and widely applicable new method for detecting highly degradable samples, which can be applied in forensic practice.

[0065] In this invention, the challenge lies in screening for multiple insertion / deletion genetic markers with good polymorphism, low recombination rate, and small core regions, and designing amplification primers with a product length not exceeding 125 bp. This invention considers the following factors:

[0066] 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;

[0067] 2) The length of the amplified fragment is between 70-125 bp, which is suitable for the detection of highly degraded samples;

[0068] 3) The primer annealing temperatures are similar;

[0069] 4) There are no obvious mismatches, hairpin structures, or dimer structures in the primers themselves, between primers, or between primers and template.

[0070] Allelic genotyping standard mixtures facilitate accurate and rapid analysis of sample genotypes. The allelic genotyping standards provided in this invention include alleles of all 26 multiple insertion / deletion genetic markers and one sex identification gene, totaling 80 fluorescently labeled fragments.

[0071] Meanwhile, this invention names the alleles of multiple insertion / deletion genetic markers. The number "10" represents the shortest allele, "12" represents the allele that is 2 bases larger than the allele named "10", "13" represents the allele that is 3 bases larger than the allele named "10", and so on.

[0072] The kit described in this invention can be used to analyze highly degraded biological samples. The specific analysis method is as follows:

[0073] (1) Extract DNA from the highly degraded sample to be tested as an amplification template;

[0074] (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 1.5 μL PCR mix, 1.0 μL multiplex amplification primer mixture, 0.5 μL DNA template, and 2.0 μL deionized water. The cycling parameters for the multiplex amplification PCR reaction were: 95℃, 2 min; 94℃, 10 s, 60℃, 80 s, 26 cycles; then 60℃, 40 min; stored at 4℃. Note: Highly degraded DNA exhibits a random effect during amplification; 3-5 parallel amplifications are required for the PCR reaction.

[0075] (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.

[0076] The following specific examples further illustrate this point. The main reagents and instruments used in this invention are as follows:

[0077] 1) Automated Laser Fluorescence Capillary Electrophoresis 3130 Genetic Analyzer, ABI Corporation;

[0078] 2) T100 TM PCR amplification instrument, BIO-RAD Corporation;

[0079] 3) High-speed centrifuges from Thermo Electron LED GmbH and ThermoFisher.

[0080] 4) NanoDrop one ultra-micro volume spectrophotometer, Thermo Scientific;

[0081] 5) Pipette, Eppendorf;

[0082] 6) Hi-Di formamide, ABI Pharmaceuticals;

[0083] 7) Molecular weight internal standard (SD550), Suzhou Qilin Intelligent Cell Biotechnology Co., Ltd.;

[0084] 8) Universal column-based DNA extraction kit, Guangzhou Meiji Biotechnology Co., Ltd.;

[0085] 9) DNA extraction kit for paraffin tissue sections, Guangzhou Meiji Biotechnology Co., Ltd.;

[0086] 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.

[0087] Example 1 Preparation of the reagent kit

[0088] The Six-Color Fluorescent Multiple Insertion / Deletion Genetic Marker Complex Detection Kit for Typing Highly Degraded Samples includes the following reagents packaged separately:

[0089] a) The multiplex amplification primer mixture is 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 pmol / μL, and then mixed according to the final concentration ratio in the amplification system shown in Table 3 to prepare the multiplex amplification primer mixture.

[0090] b) The PCR reaction mixture used was PCR mix from Suzhou Qilin Intelligent Cell Biotechnology Co., Ltd.

[0091] c) Allele typing standard mixture: composed of 80 fluorescently labeled DNA fragments, namely the amplification products of different alleles of the primer pairs in Table 2, with five colors (blue, green, black, red, and purple) fluorescence, namely FAM, HEX, TAMRA, ROX, and PURP, and an internal standard labeled with orange fluorescence.

[0092] The reagents described above are packaged according to their respective standard requirements to obtain a highly degraded sample detection kit based on multiple insertion and deletion genetic markers, which can be used for subsequent experiments.

[0093] Table 3 Concentration of Primers for Multiplex Amplification

[0094]

[0095] Example 2: Detection of artificially degraded DNA using the kit of the present invention

[0096] The artificial degradation model was created using a 2 ng / µL DNA sample (from one of 254 collected bloodstain samples from Han Chinese in Hunan) in a 100°C water bath for specific time periods (0 min, 20 min, 40 min, 60 min, and 80 min). The artificial degradation products were genotyped using the kit from Example 1 and the SureID panglobal STR kit.

[0097] Test results are shown Figures 4-7 . Figure 4 The kit from Example 1 was used to detect DNA typing results with different degrees of degradation. Figure 5The 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 7 The graph shows a comparison of the allele detection rates of the kit in Example 1 and the SureID panglobal STR kit when detecting artificially degraded DNA. It can be seen that the kit of the present invention is significantly better than the SureID panglobal STR kit when detecting highly degraded DNA, and has strong potential for detection of highly degraded samples.

[0098] Example 3: Detection of DNA from Paraffin-Embedded Tissue Using the Kit of the Present Invention

[0099] DNA was extracted from paraffin-embedded tissue (heart tissue 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 kit from Example 1 and the SureID panglobal STR kit. The results are shown in [Figure 1]. Figures 8-10 .

[0100] Figure 8 The results of blood DNA typing were obtained using the kit in Example 1. Figure 9 The results of DNA typing of paraffin-embedded tissues detected by the kit in Example 1 were compared. Both kits showed all allele peaks and the typing results were consistent, 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.

[0101] from Figures 8-10As 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 in Example 1 can still detect all 27 alleles. This indicates that the kit in Example 1 is significantly better than the conventional STR kit in obtaining complete typing from highly degraded samples in practical applications, providing a new method for typing highly degraded samples.

[0102] Example 4: Detection of Hunan Han Chinese samples and calculation of population genetic parameters using the kit.

[0103] Bloodstain samples from Han Chinese populations in Hunan Province were detected using the six-color fluorescent multiplex insertion / deletion genetic marker composite detection kit for typing highly degraded samples, as described in Example 1. The specific detection procedure is as follows:

[0104] a. The DNA extracted using a universal column-based DNA extraction kit was quantified using a Nano Drop One micro-volume spectrophotometer and then diluted to 2 ng / µL to serve as a template for multiplex amplification.

[0105] b. Using the DNA template from step a, the multiplex amplification primer mixture and multiplex amplification reaction mixture in Table 3, perform multiplex PCR amplification of the sample in the amplification system shown in Table 4.

[0106] Table 4 Amplification System

[0107]

[0108] The cycling parameters for the multiplex amplification PCR reaction were: 95℃, 2 min; 94℃, 10 s, 60℃, 80 s, 26 cycles; then 60℃, 40 min; and stored at 4℃.

[0109] c. Capillary electrophoresis

[0110] 1 µL of each amplification product and allele genotyping standard was added to 9 µL of Hi-Di formamide and 0.2 µL of SD550 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).

[0111] Using the kit from Example 1, genotyping was performed on 254 unrelated individuals of Southern Han ethnicity from Hunan Province according to the detection procedure described above, and complete alleles were obtained. Figure 3This is the genotyping result of a Han Chinese sample from Hunan. The genotyping of all 26 multiple insertion / deletion genetic markers and one sex identification gene showed no interfering peaks, and the alleles of the 27 loci in this sample could be clearly identified.

[0112] Using STRAF and Arlequin v3.5 software, the allele frequencies (Table 5) and forensic parameters (Table 6) of the above 26 multiple insertion / deletion genetic markers were calculated, including the matching probability (MP), polymorphic information content (PIC), exclusion probability (PE), typical paternity index (TPI), expected heterozygosity (He), observed heterozygosity (Ho), Hardy-Weinberg equilibrium p-value (HWE-p), and effective number of alleles (Ae).

[0113] Table 5. Allele frequencies of 26 multiple insertion / deletion loci in 254 unrelated Han Chinese individuals from southern China.

[0114]

[0115] Table 6. Forensic parameters of 26 multiple insertion / deletion loci

[0116]

[0117] Table 5 shows that all 26 multiple insertion / deletion (Multi-InDel) genetic markers were confirmed to have three distinct alleles, rather than simple binary (insertion / deletion) polymorphism. This confirms that "multiple" insertion / deletion combinations are superior to single InDels and form the basis for high discriminative power. The allele frequency distribution at most loci was relatively balanced, with no absolutely dominant alleles (such as D12MIW1 and D18MIW4), which is beneficial for improving heterozygosity and individual identification ability. Overall, all 26 multiple insertion / deletion (Multi-InDel) genetic markers showed a balanced distribution and high polymorphism in the Han Chinese population of southern China, which is highly valuable for ensuring the effectiveness of the kit system.

[0118] As shown in Table 6, the MP values ​​of the 26 loci range from 0.1882 (D12MIW1) to 0.3276 (D18MIW1), with the MP values ​​of the vast majority of loci being below 0.26. Such a low single-locus matching probability is what ultimately allows for a cumulative random matching probability as low as 10-1. -17The magnitude of the data is a fundamental guarantee. All loci have a PIC value greater than 0.4, with 20 loci (77%) having a PIC greater than 0.5, indicating high polymorphism. In particular, D12MIW1 has the highest PIC (0.5911), while D8MIM57 (0.5834) and D2MIS16 (0.5802) also show excellent polymorphism. The Ho and He values ​​for most loci are close and at a high level (mostly between 0.55 and 0.67), indicating rich genetic diversity at these loci in this population, and the observed data are consistent with the expected genetic equilibrium. High heterozygosity is a key characteristic of efficient forensic genetic markers.

[0119] Meanwhile, the probability of non-paternity exclusion (PE) is a direct indicator of the efficacy of paternity testing. In Table 6, the PE values ​​for the 26 loci range from 0.1841 (D11MIW1) to 0.4543 (D2MIS16). Despite these differences, several loci, such as D2MIS16, D12MIW1, D14MIW2, and D8MIM57, have PE values ​​exceeding 0.35, demonstrating high single-locus exclusion ability and collectively contributing to a cumulative non-paternity exclusion rate as high as 0.999925. The typical paternity index (TPI) reflects the ability of a locus to support a paternal relationship when the mother is confirmed to be heterozygous. Except for D11MIW1 (TPI=0.9922), which is slightly below 1, the TPIs of the remaining loci are all greater than 1, with loci such as D2MIS16 (1.7639) and D12MIW1 (1.5301) providing strong supporting evidence.

[0120] Table 6 also shows the good genetic quality of the 26 markers. Hardy-Weinberg equilibrium (HWE-p value): Except for D7MIW3 (p=0.0289) and D18MIW4 (p=0.0036), which deviated from equilibrium at the 0.05 level, and D11MIW1 (p=0.0516), which was close to the critical value, all were greater than the Bonferroni-corrected p value of 0.0019 (0.05 / 26). The p values ​​of the remaining 23 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 affected by factors such as obvious population substructure, selection, or mutation, making the data reliable. Effective allele count (Ae): This value directly reflects the actual contribution of allele frequency distribution to polymorphism. The Ae values ​​of all loci were greater than 2, with D12MIW1 having the highest (2.9866), further confirming its excellent polymorphism.

[0121] Example 5: Application of Triplets in Paternity Testing

[0122] 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.

[0123] Direct amplification and genotyping were performed using the kit from Example 1 to obtain complete allele typing. Figures 11-13 ).

[0124] According to the "GA / T 965-2011 Standard for Forensic DNA Paternity Testing", based on the allele frequencies obtained from the population survey of 254 unrelated Han Chinese individuals in southern China in Example 5 (see Table 5), the paternity index (PI) and cumulative paternity index (CPI) of the triad at each multiple insertion / deletion locus were calculated.

[0125] Table 7 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 9,199,855.8544, proving that the kit of Example 1 can be applied to paternity testing of highly degraded samples.

[0126] Table 7. Paternity index of the triad at 26 multiple insertion / deletion loci.

[0127]

[0128] Table 8. Nucleotide sequences of allele standards in the allele standard mixture

[0129]

[0130]

[0131]

[0132]

[0133] The sequence before the "-" symbol is a fluorescent dye label.

[0134] 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 primer set for amplifying a genetic marker system for typing highly degraded samples, characterized in that, The genetic marker system consists of primers with nucleotide sequences as shown in SEQ ID NO. 1-56, respectively; the genetic marker system comprises the Multi-InDel site located at GRCh37.p13 of the reference genome and the ameliocin gene, as shown in the table below. AMEL Composition of sex identification loci: 。 2. The application of the primer set according to claim 1 in the preparation of a reagent kit for the identification of highly degradable forensic specimens.

3. A reagent kit for the identification of highly degradable forensic samples, characterized in that, The kit includes the primer set as described in claim 1.

4. The reagent kit according to claim 3, characterized in that, The kit also includes a mixture of allele typing standards, DNA standards, and a multiplex amplification reaction mixture.

5. The application of the primer set according to claim 1 or the kit according to claim 3 or 4 in the identification of highly degraded forensic specimens.

6. The application according to claim 5, 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.

7. The application of the primer set of claim 1 or the kit of claim 3 or 4 in the identification of individuals or kinship of highly degraded samples.

8. The application according to claim 7, characterized in that, The DNA of the highly degraded biological sample to be tested is amplified by multiplex PCR using the primer set described in claim 1 or the kit described in claim 3 or 4 to obtain amplification products; the amplification products are then detected by capillary electrophoresis; based on the capillary electrophoresis results, the Multi-InDel site and amelamine gene of the biological sample to be tested are analyzed. AMEL Genotypes at sex identification loci; individual identification or kinship determination of the biological samples to be tested based on the genotype analysis results.

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