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Reagent kit for detecting gene loci on basis of next generation sequencing technologies and special primer combination of reagent kit

A technology of primer combination and sequence, applied in the field of forensic science, can solve the problems of unfavorable degradation specimen typing, does not support users' independent selection of STR loci, and expensive kits, etc.

Active Publication Date: 2017-08-29
INST OF FORENSIC SCI OF MIN OF PUBLIC SECURITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] DNA physical evidence is playing an increasingly important role in modern forensic science. At present, capillary electrophoresis (CE) is mainly used to detect the length polymorphism of each STR locus, but in CE length polymorphism Under the typing system, all alleles with the same number of nucleotides are considered to be the same allele, and it is easy to cause some heterozygous loci to be judged as homozygous due to different allele sequences but the same length, and because The capillary electrophoresis detection platform adopts multi-color fluorescence compound amplification technology. In order to arrange more loci in each color fluorescence, it is necessary to reserve longer flanking sequences for some loci when designing primers, resulting in a decrease in amplification efficiency, which is especially unfavorable Classification of degraded samples
The application of next-generation sequencing in the field of forensic science is still in its infancy, but with the increasing demand for accurate detection of difficult biological sample samples such as degraded samples in the process of handling cases, it is necessary to use next-generation sequencing technology (second generation sequencing, SGS) to provide more accurate and comprehensive test results
The above kits are expensive, and none of them support users to independently select STR loci suitable for the Chinese population. The supporting data analysis program can only analyze the sequencing data of the inherent loci in the respective kits, which has certain limitations.

Method used

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  • Reagent kit for detecting gene loci on basis of next generation sequencing technologies and special primer combination of reagent kit
  • Reagent kit for detecting gene loci on basis of next generation sequencing technologies and special primer combination of reagent kit
  • Reagent kit for detecting gene loci on basis of next generation sequencing technologies and special primer combination of reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1. Preparation of a kit for detecting loci based on next-generation sequencing technology

[0086] 1. Screening of autosomal STR loci

[0087] 1. The inventors of the present invention first screened out 50 autosomal STR loci with reference to the loci in the existing kit (such as the kit for the Illumina MiseqFGx next-generation sequencer) and the loci suitable for Chinese; Then organize the core and flanking region sequences (1000-1500bp in length) of 50 autosomal STR loci into Fasta format, and set up 9 folders, corresponding to 60bp, 80bp, 100bp, 120bp, 150bp, 200bp, 250bp, 300bp , 400bp 9 expected core fragment length gradients, each folder contains pre-arranged full-sequence FSTA files, run the GMATA Marker designing function for the FASTA files in each folder; select amplification from the primer design results A pair of primers whose product fragment length is less than 251bp, and fluorescence is added to the corresponding primers.

[0088] 2. Taking t...

Embodiment 2

[0100] Example 2. Application of the kit for detecting loci based on next-generation sequencing technology

[0101] 1. DNA sample preparation

[0102] Take the standard 2800M and dilute it with ultrapure water to obtain a standard 2800M aqueous solution with a concentration of 1ng / mL.

[0103] 2. Library preparation

[0104] 1. PCR amplification

[0105]Using the standard 2800M aqueous solution obtained in step 1 as a template and the primer mixture prepared in step 3 of Example 1 as primers, PCR amplification was performed to obtain PCR amplification products.

[0106] The reaction system is 20 μL, which consists of 10 μL Master Mix (product of the Physical Evidence Identification Center of the Ministry of Public Security), 9 μL primer mix and 1 μL standard 2800M aqueous solution. In this reaction system, the concentration of primer 3, primer 4, primer 9, primer 10, primer 17, primer 18, primer 31, primer 32, primer 43, primer 44, primer 49 and primer 50 was 0.05 μM. 2. P...

Embodiment 3

[0120] Example 3. Verification of the accuracy of the kit for detecting loci based on next-generation sequencing technology

[0121] Take 1ng of the standard product 2800M, and perform capillary electrophoresis detection according to the instructions of the GlobalFiler PCR Amplification Kit to obtain the allelic genotype of the locus. The typing results are detailed in column 2 of Table 4.

[0122] The standard product 2800M was detected according to the steps in Example 2 to obtain the allelic genotype of the locus. The typing results are detailed in column 3 of Table 4.

[0123] The results showed that the genetic locus overlapped by the next-generation sequencing technology-based detection locus kit prepared in Example 1 and the GlobalFiler PCR Amplification Kit had exactly the same typing results in the standard 2800M sample.

[0124] Table 4

[0125]

[0126]

[0127] Note: "-" indicates absence; "'" indicates different subtypes of alleles, see Table 3 for detail...

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Abstract

The invention discloses a reagent kit for detecting gene loci on the basis of next generation sequencing technologies and a special primer combination of the reagent kit. The primer combination comprises 60 types of DNA (deoxyribonucleic acid) molecules shown from sequences 1 to sequences 60 in sequence tables. The reagent kit for detecting the gene loci on the basis of the next generation sequencing technologies and the primer combination have the advantages that as proved by experiments, accurate typing results can be obtained when the reagent kit is used for detecting STR (short tandem repeat) typing of the 30 gene loci in standard substances 2800M; the reagent kit has important application value.

Description

technical field [0001] The invention relates to the technical field of forensic medicine, in particular to a kit for detecting gene loci based on next-generation sequencing technology and a special primer combination thereof. Background technique [0002] DNA physical evidence is playing an increasingly important role in modern forensic science. At present, capillary electrophoresis (CE) is mainly used to detect the length polymorphism of each STR locus, but in CE length polymorphism Under the typing system, all alleles with the same number of nucleotides are considered to be the same allele, and it is easy to cause some heterozygous loci to be judged as homozygous due to different allele sequences but the same length, and because The capillary electrophoresis detection platform adopts multi-color fluorescence compound amplification technology. In order to arrange more loci in each color fluorescence, it is necessary to reserve longer flanking sequences for some loci when de...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
CPCC12Q1/6869C12Q1/6876C12Q2600/156C12Q2600/16C12Q2535/122C12Q2537/143
Inventor 王乐季安全叶健赵兴春刘毅成武波
Owner INST OF FORENSIC SCI OF MIN OF PUBLIC SECURITY
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