A compound amplification system for analyzing micro-haplotypes of degraded samples and its construction method

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

Active Publication Date: 2021-07-09
SHANXI MEDICAL UNIV
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  • Description
  • Claims
  • Application Information

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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

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  • A compound amplification system for analyzing micro-haplotypes of degraded samples and its construction method
  • A compound amplification system for analyzing micro-haplotypes of degraded samples and its construction method
  • A compound amplification system for analyzing micro-haplotypes of degraded samples and its construction method

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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 sites and the frequency corresponding to each type;

[0078] ④Select 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 above selected sites in 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 resu...

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 amplified from these sites by PCR and the PCR products in the human genome Feb.2009 (GRCh37 / hg19 version) Position in the reference genome. Wherein, 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 Tabl...

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 product, use an agarose gel recovery kit to purify, and after the library inspection is qualified, use a MiSeq sequencer for sequencing.

[0120] (4) Perform quality control on the fastq file obtained by sequencing, convert the fastq fi...

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Abstract

The invention provides a micro-haplotype composite amplification system in the analysis of degraded sample materials, a construction method thereof and related haplotype frequencies. The present invention provides a group of genetic marker compositions totaling 23 micro-haplotypes and their rs numbers. According to the amplification primers of 23 micro-haplotypes, a complex amplification system of micro-haplotypes for the analysis of degraded samples was constructed. Also provided herein is a kit comprising amplification primers for the aforementioned 23 micro-haplotypes. The micro-haplotype complex amplification system provided by the invention can carry out forensic identification on degraded samples, and has high sensitivity and strong identification ability.

Description

technical field [0001] The invention belongs to the field of forensic medicine, and in particular relates to a micro-haplotype complex amplification system in the analysis of degraded specimens, 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 nucleotide po...

Claims

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Application Information

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Patent Type & Authority Patents(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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