Six-color fluorescence STR parting method for synchronously detecting 28 gene loci and special kit of method

A gene locus, D21S11 technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as development lag, late start, and difficulty in accommodating the number of loci

Pending Publication Date: 2019-07-30
BGI FORENSIC TECH (SHENZHEN) CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Compared with foreign countries, China started late and its development is relatively lagging behind. In China, this technical field is still dominated by 5-color fluorescent labels, which is difficult to accommodate more sites, and most kits are based on the early 13 CODIS sites. Add a few loci suitable for the domestic population, such as the Goldeneye 20A (20 loci) of the basic cognitive company, the MicroReaderTM 21D (21 loci) of the micro gene, and the AGCU Expressmarker 22 ( 22 sites) and STRtyper-21G (21 sites) from Ningbo Haiers Gene Technology Co., Ltd.

Method used

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  • Six-color fluorescence STR parting method for synchronously detecting 28 gene loci and special kit of method
  • Six-color fluorescence STR parting method for synchronously detecting 28 gene loci and special kit of method
  • Six-color fluorescence STR parting method for synchronously detecting 28 gene loci and special kit of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1, the preparation of the compound amplification system based on 28 gene loci

[0045] 1. Screening of 28 loci

[0046] The inventors of the present invention, in order to meet the requirements for the amount of information, individual recognition and compatibility of the detection of STR gene loci, used six-color fluorescent markers to increase the number of accommodated loci, and at the same time increased the compatibility of the kit loci. It can cover the human DNA databases of most countries in the world. The inventors of the present invention finally screened a total of 28 gene loci including 24 autosomal STR loci, 1 sex identification locus AMEL, 1 indel locus Yindel and 2 sex chromosome STR loci. 24个常染色体STR基因位点为D3S1358、D13S317、D7S820、D16S539、SE33、D10S1248、D5S818、D21S11、TPOX、D1S1656、D6S1043、D19S433、D22S1045、D8S1179、Penta E、D2S441、D12S391、D2S1338、vWA、Penta D , TH01, D18S51, CSF1PO and FGA. The two sex chromosome STR loci are DYS391 and DXS6795.

[0...

Embodiment 2

[0106] Example 2, the typing detection of DNA standard 9947A and DNA standard 9948 based on the multiple amplification system of 28 gene loci prepared in Example 1

[0107] DNA standard product 9947A stock solution (concentration: 10ng / μL) and DNA standard product 9948 stock solution (concentration: 10ng / μL) are both products of Xinhai Biotechnology Co., Ltd.

[0108] 1. Acquisition of DNA Standard Diluent

[0109] Take 1 μL DNA standard stock solution (DNA standard 9947A stock solution or DNA standard 9948 stock solution), and add 9 μL TE buffer to obtain a DNA standard dilution (concentration: 1 ng / μL).

[0110] 2. Take 15 μL of the complex amplification system based on 28 gene loci prepared in step 4 of Example 1, add 0.4 μL of hot-start Taq enzyme (including 2 U), 1 μL of DNA standard diluent and 8.6 μL of nuclease-free water, Get the reaction system.

[0111] 3. Put the reaction system obtained in step 2 into a Bioer G-1000 thermal cycler for PCR amplification to obtain...

Embodiment 3

[0124] Example 3, the typing detection of a triplet family based on the multiple amplification system of 28 gene loci prepared in Example 1

[0125] The blood samples of the triplet family were provided by Guangdong Huada Forensic Evidence Identification Institute.

[0126] 1. Obtaining genomic DNA from family blood samples

[0127] Genomic DNA was extracted from the blood samples of triplet families by chelex-100 method.

[0128] 2. Take 15 μL of the multiplex amplification system based on 28 gene loci prepared in step 4 of Example 1, add 0.4 μL of hot-start Taq enzyme (including 2 U), 1 μL of genomic DNA from family blood samples and 8.6 μL of nuclease-free water , to get the reaction system.

[0129] 3. Put the reaction system obtained in step 2 into a Bioer G-1000 thermal cycler for PCR amplification to obtain PCR amplification products. See Table 7 for the temperature cycle conditions of the Bioer G-1000 thermal cycler.

[0130] 4. After completing step 3, mix 9.5 μL ...

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Abstract

The invention discloses a six-color fluorescence STR parting method for synchronously detecting 28 gene loci and a special kit of the method. The method comprises the following steps that a genome DNAof a to-be-detected individual is used as a template, the kit is adopted for PCR amplification, and a PCR amplification product is obtained; the PCR amplification product is taken, subjected to degeneration and then subjected to capillary electrophoresis detection, and genotypes of the 28 gene loci of the genome DNA of the to-be-detected individual are obtained; according to the genetypes of the28 gene loci, an STR parting result of the to-be-detected individual is obtained. The kit comprises a primer pair combination, nucleotide sequences of 56 primers forming the primer pair combination are shown in the sequences 1-56 in a sequence table in sequence, and one primer in each primer pair is marked with fluorescence. The kit is compatible with human DNA database standard allele loci of many countries, suitable for the Chinese nation population and more suitable for many countries in the whole world. The kit has high application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a six-color fluorescent STR typing method for synchronous detection of 28 gene sites and a special kit thereof. Background technique [0002] Short tandem repeats (short tandem repeats, STRs) are a class of molecular genetic markers that widely exist in eukaryotic genomes and belong to the second generation of genetic markers. In the human genome, there is one STR site about every 15-20kb, and they account for 10% of the total human genome. These STRs usually consist of tandem repeating units of 2-6 bases. Due to the difference in repeating units and repeating times, they show large differences among people of different races and regions, which is manifested as genetic polymorphism. Compared with other genetic markers, STR genetic markers not only have a large amount of information, but also have large differences among different individuals, high polymorphism, small fra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11
CPCC12Q1/6876C12Q1/686C12Q2600/16C12Q2563/107C12Q2537/143C12Q2565/125
Inventor 李生斌伏东科熊文娟李波
Owner BGI FORENSIC TECH (SHENZHEN) CO LTD
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