CN-122012741-A - 44 InDel genetic marker system for highly degraded sample typing, detection primer and application thereof

Abstract

The invention discloses 44 InDel genetic marker systems for highly degraded sample typing, a detection primer and application thereof, and belongs to the technical field of medical identification. The invention is verified by bioinformatics screening and experiments, constructs an InDel (InDel) genetic marker system with the length of the amplicon not exceeding 125 bp, which consists of 44 autosomal InDel and sex identification gene AMEL loci, is suitable for capillary electrophoresis typing and is specially designed for highly degrading detection materials. The system is simple and convenient to operate, economical and efficient, can obviously improve the complete parting success rate of the degraded sample, and is convenient to popularize and apply in a forensic laboratory. The invention provides a novel technical scheme of high-efficiency compatible capillary electrophoresis platform for forensic practice.

Inventors

• LI JIENAN
• ZHALAGABAIYILA
• WANG YUEPENG
• CAI JIFENG
• Xie Qiheng
• WEN DAN
• QU WEIFENG

Assignees

• 中南大学

Dates

Publication Date

20260512

Application Date

20260316

Claims (10)

1. 1. A genetic marker system for the genotyping of highly degraded samples, characterized in that it consists of the InDel site located in the reference genome grch37.p13 as shown in the following table: 。
2. 2. A primer set for amplifying the genetic marker system of claim 1, comprising primers having nucleotide sequences set forth in SEQ ID nos. 1-88, respectively.
3. 3. Use of the primer set of claim 2 in the preparation of a kit for forensic identification.
4. 4. A kit for forensic identification, comprising the primer set of claim 2.
5. 5. The kit of claim 4, further comprising a primer pair for amplifying AMEL sex identification sites of the amelogenin gene.
6. 6. The kit of claim 4, further comprising an allelic typing standard mixture, a DNA standard, and a multiplex amplification reaction mixture.
7. 7. Use of the primer set of claim 2 or the kit of any one of claims 4-6 in forensic high degradation assay identification.
8. 8. The use of claim 7, wherein the highly degraded test material is selected from at least one of a large-scale catastrophic event biological test material, old bone, formalin-fixed paraffin-embedded tissue, and non-follicular hair.
9. 9. Use of a primer set according to claim 2 or a kit according to any one of claims 4 to 6 for identification of individuals or for genetic relationship identification.
10. 10. The use according to claim 9, wherein the primer set according to claim 2 or the kit according to any one of claims 4 to 6 is used for multiplex PCR amplification of DNA of a biological test material to be tested to obtain an amplification product, the amplification product is subjected to capillary electrophoresis detection, the genotype of the InDel locus of the biological test material to be tested in claim 1 is analyzed according to the result of capillary electrophoresis, and the biological test material to be tested is subjected to individual identification or genetic relationship identification according to the result of analysis of the genotype.

Description

44 InDel genetic marker system for highly degraded sample typing, detection primer and application thereof Technical Field The invention relates to the technical field of forensic identification, in particular to a 44 InDel genetic marker system for highly degraded sample typing, a detection primer and application thereof. Background The highly degraded detection material is a difficult detection material frequently encountered in forensic practice and is also a difficulty in forensic research. When the biological evidence is subjected to high temperature, humidity, ultraviolet radiation, microorganisms, strong acids and strong bases, long-chain DNA molecules are decomposed into small DNA fragments. If the DNA molecule in the detection material is extremely severely fragmented, the length is even less than 150bp, the detection material can be called as a highly degraded detection material. Such inspection materials are commonly found in bursless hair shafts, formalin-fixed paraffin embedded samples, mature table former practice inspection materials, and highly spoiled inspection materials in large scale disaster events such as air spaces, earthquakes, tsunamis, and the like. Short Tandem Repeats (STRs) are the most widely used genetic markers in forensic applications, but are not ideal genetic markers for analysis of highly degraded test materials by forensics due to their relatively large amplicon size and concomitant shadow peaks (stutter peaks) in typing. Even if the miniSTR system is specially designed for degradation samples, the amplified fragments of the miniSTR system are between 100 and 330bp, and the miniSTR system is often not provided with parting results when being used for high degradation detection material identification. The concept of a micro-haplotype genetic marker (Microhaplotype) proposed by k.k. Kidd in 2014, which refers to a sequence containing 2 or more SNPs in a short fragment and having a haplotype polymorphism, is considered as an ideal genetic marker for detecting degradation specimens. Although micro haplotypes have the advantages of low mutation rate, short amplicon length, no occurrence of false peaks such as shadow peaks during typing, and the like, detection of the micro haplotypes depends on a second generation sequencing (MPS) platform, instrument equipment and consumable materials are expensive, the operation is complex, the detection period is long, professional technicians are required, and the defects of high requirement on template DNA quality and the like also make the micro haplotypes not widely developed in a forensic DNA laboratory. InDel polymorphism-InDel (insertion/deletion) is a DNA polymorphism due to insertion or deletion of a DNA fragment. Compared with other molecular genetic markers, inDel has the characteristics that 1) InDel genetic markers widely exist in human genome, average 7.2 kb has one InDel, the number of the InDel genetic markers is inferior to that of the first SNP, 2) the single InDel locus has short fragments and is suitable for degradation detection materials, 3) the InDel has low mutation rate of about 10 -8 and rarely generates repeated mutation, the InDel is suitable for crowd genetic polymorphism analysis, 4) the InDel fragments have lengths of one to hundreds of single nucleotides and are expressed as length polymorphism, the typing detection can be carried out by utilizing a capillary electrophoresis (CAPILLARY ELECTROPHORESIS, CE) platform, the application in basic-level forensic laboratories is guaranteed, and 5) the InDel genetic markers can also be sequenced by utilizing a large-scale parallel sequencing platform and have very strong potential application and research value. Therefore, inDel has the advantages of STR and SNP, is an emerging molecular genetic marker with strong forensic scientific research and application value, and is suitable for basic forensic laboratories, and the characteristics make InDel an ideal genetic marker for high degradation detection material identification. Currently, limited by the selection of target amplified fragment length and InDel sites, the number of loci that can be accommodated in a single reaction system is limited, the amount of genetic information that can be covered is limited, and existing studies are limited by amplicon length or the number of genetic markers in a multiplex amplification system, which makes it difficult to accurately detect highly degraded samples. Therefore, a group of composite amplification detection systems with higher identification efficiency and suitable for detecting the highly degraded detection materials are developed, and the method has important significance in the forensic actual detection. Disclosure of Invention The invention aims to provide a 44 InDel genetic marker system for highly degrading sample typing, a detection primer and application thereof, so as to solve the problems in the prior art. The invention provides a set of compound detection sy