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Microhaplotype multiplex amplification system in mixed sample analysis and construction method thereof and related haplotype frequency

A compound amplification system and haplotype technology, applied in the field of forensic medicine, can solve problems such as cumbersome operations, and achieve the effects of high sensitivity, consistent amplification efficiency, and high system efficiency.

Active Publication Date: 2020-11-06
SHANXI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the number of amplifications in this technical scheme is divided into three times, and the operation is cumbersome and needs to be further optimized.

Method used

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  • Microhaplotype multiplex amplification system in mixed sample analysis and construction method thereof and related haplotype frequency
  • Microhaplotype multiplex amplification system in mixed sample analysis and construction method thereof and related haplotype frequency
  • Microhaplotype multiplex amplification system in mixed sample analysis and construction method thereof and related haplotype frequency

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Embodiment 1 Construction of micro-haplotype compound amplification system

[0072] (1) Site selection:

[0073] ①Download 82 Chinese SNP sites with rs numbers on chromosomes 1-22 from the hapmap database;

[0074] ②According to the position, select the fragment within the range of 250bp, containing 3 or more SNP sequences;

[0075] ③Use haploview software to calculate the haplotypes of these loci and the frequency corresponding to each type;

[0076] ④ Select the site with more than 4 micro-haplotypes at a specific position, and the probability of each type is relatively close;

[0077] ⑤ Put the above selected loci in the Chinese population database of BGI to calculate the frequency, and select the loci whose allele frequency is greater than or equal to 0.1.

[0078] (2) Primer design: PCR multiplex amplification is required in the follow-up work of the selected sites above, and primer design for multiplex multiplex amplification PCR needs to be considered: the len...

Embodiment 2

[0081] Embodiment 2 A kind of micro haplotype composite amplification system

[0082] 1. A micro-haplotype complex amplification system obtained according to the construction method in Example 1.

[0083] 1.1 Micro-haplotype loci

[0084] 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 length of the PCR amplification product is within the range of 250bp, and the SNPs included in the micro-haplotypes are all 4 or more.

[0085] Table 4 Micro-haplotype target fragment length, position in hg19 reference genome and rs number

[0086]

[0087] 1.2 Amplification primers in the multiplex amplification system

[0088] In the composite amplification system, the forward and reverse primers on each chromosome of the 18 micro-haploty...

Embodiment 3

[0112] Embodiment 3 Han / Tibetan / Uyghur 3 groups 50 sample haplotype frequency results

[0113] Since the micro-haplotypes provided by the present invention are all newly designed, there is no corresponding frequency information that can be used to analyze mixed samples when applied to the kit, so the frequency of these micro-haplotypes needs to be calculated first.

[0114] (1) DNA extraction: using Qiagen The DNA Investigator Kit kit was used to extract the whole genome DNA of the samples.

[0115] (2) Perform multiplex PCR amplification on the extracted DNA according to the multiplex amplification system provided in Example 2.

[0116] (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.

[0117] (4) Carry out quality control on the fastq files obtained by sequencing, convert the fastq files after quality control into fasta file...

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Abstract

The invention provides a construction method of a microhaplotype multiplex amplification system in mixed sample analysis and related haplotype frequency. The invention provides a group of genetic marker combinations, in total of 18 microhaplotypes and rs numbers of the 18 microhaplotypes . According to the 18 microhaplotype amplification primers, a microhaplotype composite amplification system formixed sample analysis is constructed. Also provided herein is a test kit comprising amplification primers of the aforementioned 18 microhaplotypes. The microhaplotype multiplex amplification system provided by the invention can be used for carrying out forensic medicine identification on a mixed test material, and is high in sensitivity and strong in identification 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 for the analysis of mixed specimens, a construction method of the micro-haplotype composite amplification system and the haplotype frequency thereof. Background technique [0002] Since the advent of DNA analysis technology, the development of more accurate, stable, informative and easy-to-detect polymorphic sites in human DNA has been a hot spot in the research of forensic evidence. Currently commonly used DNA typing techniques mainly rely on polymorphic genetic markers such as short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs). [0003] Among them, STR has developed into the mainstream genetic marker used in forensic DNA typing because of its high sensitivity, high discrimination ability, high species specificity, high accuracy of results, and easy standardization. However, due to the "tandem repe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6879C12Q1/6858G16B25/20G16B20/20
CPCC12Q1/6858C12Q1/6879C12Q1/6888C12Q2600/156C12Q2600/16C12Q2600/172G16B20/20G16B25/20C12Q2531/113C12Q2565/125C12Q2537/143
Inventor 严江伟石林玉张更谦王萌春程凤李万婷张家榕张晓梦
Owner SHANXI MEDICAL UNIV
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