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Microhaplotype composite amplification system in degradation sample analysis, and construction method thereof

A technology of compound amplification system and construction method, applied in the field of haplotype frequency, can solve the problems of STR typing technology can not obtain ideal typing results, allele loss, etc., and achieve the effect of high sensitivity and strong identification ability

Active Publication Date: 2020-12-11
SHANXI MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Chinese patent 201910328532.0 discloses a compound amplification system based on 21 micro-haplotype sites, the purpose of which is to solve the problem that the existing detection method STR typing technology cannot obtain ideal typing results, and the possibility of too many SNP sites may occur. Technical issues with bit loss

Method used

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  • Microhaplotype composite amplification system in degradation sample analysis, and construction method thereof
  • Microhaplotype composite amplification system in degradation sample analysis, and construction method thereof
  • Microhaplotype composite amplification system in degradation sample analysis, and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Embodiment 1 Construction of micro-haplotype compound amplification system

[0074] (1) Site selection:

[0075] ①Download 82 Chinese SNP sites with rs numbers on chromosomes 1-22 from the hapmap database;

[0076] ②According to the position, select the fragment within the range of 140bp, containing 3 or more SNP sequences;

[0077] ③ Haploview software was used to calculate the haplotypes of these loci and the frequency corresponding to each type;

[0078] ④ Select the site with more than 4 micro-haplotypes at a specific position, and the probability of each type is relatively close;

[0079] ⑤ Put the selected sites above into the Chinese population database of BGI to calculate the frequency, and select the sites with an allele frequency greater than or equal to 0.1.

[0080] (2) Primer design: the length of the primers is between 18-25bp; the Tm value of the primers at each locus should be as consistent as possible, and the Tm value mainly depends on the design re...

Embodiment 2

[0082] Embodiment 2 A kind of micro haplotype composite amplification system

[0083] 1. Obtain a micro-haplotype complex amplification system according to the construction method in Example 1.

[0084] 1.1 Micro-haplotype loci

[0085] Table 4 shows the number of SNP sites selected on each chromosome and the corresponding rs numbers, as well as the length of the amplified fragments and PCR products amplified from these sites by PCR in the human genome Feb.2009 (GRCh37 / hg19 version) Position in the reference genome. Among them, the amplification length of PCR is all in the range of 140bp, and the included SNPs are all between 3-4.

[0086] Table 4 Micro-haplotype amplification length, position and rs number in the hg19 reference genome

[0087]

[0088]

[0089] 1.2 Amplification primers in the multiplex amplification system

[0090] In the composite amplification system, the forward and reverse primers on each chromosome of the 23 micro-haplotypes are shown in Table...

Embodiment 3

[0115] Example 3 Haplotype frequency results of 50 irrelevant samples from 3 populations of Han / Tibetan / Uighur

[0116] Since the micro-haplotypes provided by the present invention are all new designs, there is no corresponding frequency information that can be used to analyze the degraded samples when they are applied to the kit. Therefore, the frequencies of these micro-haplotypes need to be calculated first.

[0117] (1) DNA extraction: using Qiagen The DNA Investigator Kit kit was used to extract the whole genome DNA of the samples.

[0118] (2) Perform multiplex PCR amplification on the extracted DNA according to the multiplex amplification system provided in Example 2.

[0119] (3) After performing gel electrophoresis on the obtained PCR products, use an agarose gel recovery kit to purify them, and after passing the library inspection, use a MiSeq sequencer for sequencing.

[0120] (4) Carry out quality control on the fastq files obtained by sequencing, convert the fast...

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Abstract

The invention provides a microhaplotype composite amplification system in degradation sample analysis, a construction method thereof and related haplotype frequency. The invention provides 23 microhaplotypes in total and rs numbers thereof in one group of genetic marker compositions. According to amplification primers of the 23 microhaplotypes, a microhaplotype composite amplification system for analyzing degradation samples is constructed. The invention also provides a kit comprising the amplification primers of the 23 microhaplotypes. The microhaplotype composite amplification system provided by the invention can be used for forensic identification of the degradation samples, and is high in sensitivity and strong in recognition capability.

Description

technical field [0001] The invention belongs to the field of forensic medicine, and in particular relates to a micro-haplotype composite amplification system in the analysis of degraded sample materials, a construction method thereof, and related haplotype frequencies. Background technique [0002] Forensic physical evidence is mainly to solve the problem of personal identification in judicial practice. Personal identification is based on the same identification theory as the guiding principle, and scientifically identifies the genetic markers of on-site samples. Therefore, new, more stable, accurate, fast, and informative The development and utilization of a large number of human genetic markers has always been a hot spot in forensic research. [0003] With the development of DNA analysis technology and its application in forensic science, people have gradually developed personal identification detection based on short tandem repeat (STR) length polymorphism and single nucl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6858C12N15/11G16B25/20G16B40/00G16B50/00
CPCC12Q1/6858C12Q1/6888C12Q2600/156C12Q2600/172G16B25/20G16B40/00G16B50/00C12Q2531/113C12Q2565/125
Inventor 严江伟王萌春张更谦石林玉程凤李万婷张君漆小琴杨婷婷
Owner SHANXI MEDICAL UNIV
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