str type standard substance

A technology for typing reference materials and reference materials, applied in the field of forensic DNA testing and analysis, which can solve the problems of heavy workload, high cost, and difficulty in obtaining

Active Publication Date: 2011-12-21
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

The above reference materials have some disadvantages. One is that the genotype is possessed by natural persons; the other is that the establishment of cell lines is required, which is difficult to obtain, heavy workload, complicated procedures and high cost.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1、1

[0031]Embodiment 1, the acquisition of 19 locus alleles

[0032] 1. Locus CSF1P0 alleles (N=12 (TAGA) 12 ) of the acquisition

[0033] 1. Design of primers

[0034] On the basis of determining the flanking sequence of the CSF1P0 gene locus, the primers were designed as follows: Forward primer: TGGCAGAAGCCGGAGGTAA; Reverse primer: CCGATGAGCTGCTGCCTTG

[0035] 2. PCR amplification

[0036] Using the above primer pairs, PCR amplification was carried out using the extracted human saliva genomic DNA as a template. The PCR system and reaction parameters are as follows:

[0037] a. PCR amplification system (10 μL):

[0038]

[0039] b. PCR thermal cycle parameters:

[0040] 95℃11min; 94℃1min, 60℃1min, 72℃1min, 30cycles; 60℃60min; 25℃Hold. (The annealing temperature varies according to the Tm value of different loci.)

[0041] After electrophoresis, recovery, and purification, the PCR product fragments were directly inserted into the carrier T vector (purchased from Promega...

Embodiment 2

[0164] The preparation of the standard substance of embodiment 2, STR typing

[0165] The 31 recombinant cloning vectors prepared in Step 1 to Step 19 in Example 1 were gradually mixed. With the increase of allelic DNA fragments, due to the competition mechanism inside the DNA molecule, there was a certain degree of relative balance control of each allelic fragment. It is necessary to continuously adjust the recombinant plasmids with different allelic DNA fragments to the optimal ratio, and finally establish the relative balance among multiple DNA fragments through repeated experiments, and finally form a DNA standard substance that meets the requirements. Among them: the recombinant cloning vector V-CSF1 among the 31 recombinant cloning vectors: the recombinant cloning vector V-1338-1: the recombinant cloning vector V-1338-2: the recombinant cloning vector V-1358-1: the recombinant cloning vector V-1358- 2: Recombinant cloning vector V-818: Recombinant cloning vector V-1043-1...

Embodiment 3

[0167] Embodiment 3, verification of reference material

[0168] The amplification system in the amplification experiment from step 1 to step 4 is 10ul.

[0169] 1. Typer TM 15 kit amplification

[0170] Using the standard substance actually prepared in Example 2 as a DNA template, Typer TM 15 Fluorescence multiplex amplification kit (Ministry of Public Security Evidence Identification Center) was used for amplification according to the operating instructions. Its DNA test profile is as follows figure 1 As shown, the spectrum is clear and the peak shape is sharp, which is consistent with the preset results.

[0171] 2. Identifier TM kit amplification

[0172] Using the standard substance actually prepared in Example 2 as a DNA template, the Identifilier TM Fluorescence multiplex amplification kit (ABI Company, USA) was used to amplify according to the operating instructions. The amplification parameters were set as 95°C, 5min; 94°C for 1min, 59°C for 1min, 72°C for 1mi...

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Abstract

The invention discloses a short tandem repeat (STR) parting standard substance, which is a deoxyribonucleic acid (DNA) mixture and comprises 31 DNA segments with the nucleotide sequences as 1) to 31). In the invention, all allelic genes in the bits of Chinese people groups are researched and studied through focusing on the discovered STR gene bits in the existing forensic molecular genetics, and in addition, the biostatistics technology is used for studying the gene frequency distribution of each allelic gene. On the basis, the experiments techniques such as molecular genetics, molecule biology, cell biology, genetic engineering and the like are integrally utilized, the allelic gene DNA information base is developed and built for the first time, all of the allelic gene DNA segments such as STR bits are included, further, the forensic DNA standard reference system independently developed by China is further built, and the special artificially synthesized STR standard substance is formed.

Description

technical field [0001] The invention relates to an STR typing standard substance in the field of forensic DNA testing and analysis. Background technique [0002] Short tandem repeats (short tandem repeats, STRs), also known as simple sequence repeats (SSRs), are a type of genetic marker widely distributed in the human genome, and their core sequences generally consist of 2 to 6 bases , different numbers of repeat units in the core sequence lead to different alleles in the same locus. The vast majority of STR sequences are distributed in the non-coding regions of the genome, the core repeat sequences are arranged in tandem, and the amplified fragments are generally below 400bp. Currently, they are widely used in forensic personal identification and paternity testing. [0003] With the development of forensic DNA testing and analysis technology, its testing technology has gone through three main stages: restriction fragment length polymorphism (RFLP), short tandem repeat (STR...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/63C12Q1/68
Inventor 赵兴春叶健
Owner INST OF FORENSIC SCI OF MIN OF PUBLIC SECURITY
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