Mitochondria SNP (single nucleotide polymorphism) fluorescence-labeling multiple amplification kit and application thereof

A technology of fluorescent labeling and multiple amplification, which is applied in the field of life, can solve the problems of cumbersome operation steps, expensive digestive enzymes, and heavy workload, and achieve the goal of optimizing multiple PCR systems, improving inspection speed and efficiency, and strong sample adaptability Effect

Active Publication Date: 2014-07-02
SHANG HAI GENE BIO TECH +2
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  • Abstract
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AI Technical Summary

Problems solved by technology

However, this method also has obvious limitations: the digestive enzymes used for partial purification are expensive; the operation steps are cumbersome and the workload is heavy; the success rate of sites with unknown mutations in the primer binding region is low, which is specifically reflected in the successful detection of sites in the mitochondrial control region low rate
[0005] Mitochondrial DNA research patents almost focus on the discussion of mitochondrial diseases, but rarely involve maternal identification. The only one "A Method and Application for Detecting Haplotypes of Mitochondrial Genes" uses oligonucleotides The acid probe method, the real-time quantitative detection method has high sensitivity and good specificity, but at the same time has the disadvantages of low throughput, complicated primer and probe design, etc., and is not suitable for large sample detection

Method used

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  • Mitochondria SNP (single nucleotide polymorphism) fluorescence-labeling multiple amplification kit and application thereof
  • Mitochondria SNP (single nucleotide polymorphism) fluorescence-labeling multiple amplification kit and application thereof
  • Mitochondria SNP (single nucleotide polymorphism) fluorescence-labeling multiple amplification kit and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1 Selection, grouping, primer design and establishment of reaction system of mitochondrial SNP sites of the present invention

[0039] 1. SNP site screening

[0040] Referring to the mitochondrial phylogenetic tree (mtDNA tree Build16 (19Feb2014)), combined with the genetic characteristics of the mitochondrial DNA of the Chinese population, the characteristic sites of the main mitochondrial gene haplotype groups and their subtypes were screened out; according to the mtDNA haplotype groups of the Chinese population The type tree combined with the polymorphism investigation of mitochondria in the Chinese population, literature research, and database analysis finally screened out 61 mtDNA characteristic sites, all of which have high GD values, covering the main mtDNA haplogroups and their main mtDNA haplogroups in the Chinese population. Subtype. Among them, the GD value of 73.7% of the sites is above 0.2, and the maximum value reaches 0.5. Guarantee the identifi...

Embodiment 2

[0083] Example 2 The accuracy verification and large sample test of the mitochondrial SNP detection kit of the present invention

[0084] Using the primers, grouped fluorescent labels, and amplification system and parameters in Example 1, a large sample of mitochondrial SNP detection was carried out. A total of 200 Han and 200 Uygur population samples were randomly selected, and the samples were extracted and amplified with a kit. The results of electrophoresis test showed that the main typing results of the Han nationality were D4*, B5, D5a, and D4b2, which accounted for 7%, 6.5%, 4.5%, and 4.5% respectively. Similarly, in the Uighur population, D4*, T, and W accounted for 7%, respectively. 7.5%, 7%, and 6%. As the descendants of the ancient Huaxia nationality, the Han nationality's diffusion distribution conforms to the Demic diffusion model. During the population explosion period, there was gene exchange between it and multiple ethnic groups; for Uyghurs, their typing has ...

Embodiment 3

[0085] Example 3 Detection of mtDNA SNP site heterogeneity by the kit provided by the present invention

[0086] The existence of mtDNA heterogeneity brings uncertainty to the identification of the same maternity in forensic medicine, and corresponding evaluation should be carried out in actual cases. The International Society for Forensic Genetics (ISFG) stipulates that if there is a base difference between two samples, and this base position is recognized as a heterogeneity hotspot, it cannot be ruled out; when two bases are encountered When the base positions are different, if two positions are heterogeneous hotspots, it cannot be ruled out, otherwise it cannot be judged; when three positions are different, an exclusion conclusion can be made. The kit provided by the invention can not only realize the detection of general SNP sites, but also can successfully detect when the sites have heterogeneity. In the actual sample detection, it was found that some of the 200 Uyghur s...

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Abstract

The invention relates to a mitochondria SNP (single nucleotide polymorphism) fluorescence-labeling multiple amplification kit capable of detecting 61 SNP loci at the same time. According to a mitochondria phylogenetic tree, based on genetic characteristics of Chinese populatio, 61 SNP loci which are high in polymorphism, low in mutation rate and strong in parting capacity are selected, wherein the 61 SNP lotuses comprise 54 loci in a coding region, 7 loci in a control region; the kit can be used for detecting the 61 SNP loci by virtue of specific primers. The kit which is used as a mitochondria detecting system is capable of carrying out amplification in three tubes at the same time and performing electrophoresis at the same time, so that haplotype diversity reaches 98.6%, and therefore, the mitochondria SNP fluorescence-labeling multiple amplification kit can be used as a kit for identifying the same maternal line, after autosome STR and Y chromosome STR.

Description

technical field [0001] The present invention belongs to the technical field of biological in vitro diagnosis, and embodies the joint application of the third-generation genetic marker SNP (single nucleotide polymorphism, single nucleotide polymorphism) and fluorescence detection technology, which can quickly determine the sample by using a relatively small amount of DNA template mtDNA haplotype groups, suitable for mitochondrial DNA maternal identification. Background technique [0002] At present, the kits used for individual identification in forensic science are all for the detection of autosomal STR genetic markers. Since there is no linkage relationship between them and there is no association between alleles of different genetic markers, the total matching probability of autosomes for personal identification is The product of the matching probabilities of each genetic marker, so it is not necessary to increase genetic markers indefinitely during individual identificati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/68G01N21/64C12Q1/6858C12Q2537/143C12Q2563/107C12Q2565/125
Inventor 郑卫国焦海涛周怀谷卢青杨海峰葛海鹏郭育林葛斌文
Owner SHANG HAI GENE BIO TECH
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