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Method for detecting polymorphism of 12 loci of human mitochondrial cytochrome b genes simultaneously

A cytochrome, mitochondrial technology, applied in the field of genetic engineering

Inactive Publication Date: 2010-10-06
GENERAL HOSPITAL OF PLA
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Problems solved by technology

Studies have found that Cytb 15497 mutation can cause severe obesity in Japanese, but not in Italian Caucasians (Liguori R, et al..The mtDNA 15497G / A polymorphism in cytochrome b in severe obese subjects from S outhernItaly.Nutr Metab Cardiovasc Dis.2006 Oct;16(7):466-70), suggesting that the mitochondrial phenotype has racial differences, so it is not enough to rely solely on the mitochondrial SNP differences reported in foreign literature, and it is necessary to find Chinese Population-specific SNPs

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  • Method for detecting polymorphism of 12 loci of human mitochondrial cytochrome b genes simultaneously
  • Method for detecting polymorphism of 12 loci of human mitochondrial cytochrome b genes simultaneously
  • Method for detecting polymorphism of 12 loci of human mitochondrial cytochrome b genes simultaneously

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[0060] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Example 1 The polymorphisms at 12 sites of the cytb gene were detected simultaneously in 24 Chinese cases using the method described in the present invention.

[0061] (1) Primers designed to amplify the full-length DNA fragment containing the cytochrome b gene:

[0062] Forward primer: 5'-CCATCGCTAACCCCACTAAA-3';

[0063] Reverse primer: 5'-TGGTACCCAAATCTGCTTCC-3'.

[0064] (2) Design extension primers for detecting 12 sites of the cytochrome b gene respectively, and mark the 12 extension primers with different specific sequence tags (see Table 1 and sequences 3-14).

[0065] (3) The whole gene of cytochrome b was amplified by PCR.

[0066](4) Perform single base extension reaction and perform fluorescent labeling.

[0067] (5) Hybridization, add 8 μL of hybridization solution (50 μL of hybridization reagent to 850 μL of hybridizati...

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Abstract

The invention relates to a method for detecting the polymorphism of 12 loci of human mitochondrial cytochrome b genes simultaneously, and provides a method for detecting the polymorphism of 12 loci of mitochondrial cytochrome b genes (Cytochrome b, Cytb) simultaneously in the same system, which realizes the detection of the polymorphism of the 12 loci of the cytochrome cytb genes simultaneously in the same reaction system by combining the technology of polymerase chain reaction (PCR) with the technology of single basic group extension and fluorescence microarray hybridization. The fluorescence intensity and fluorescence type which are generated by single basic group extension only correspond to a genotype, and a polymorphic locus has only three kinds of characteristic fluorescence at most, and thus the genotypes of samples corresponding to all the same peak types can be obtained only by one-time DNA sequencing verification. Compared with a detection method by direct sequencing, the method of the invention has short time, namely experiments are finished within one day, and has high flux, namely the 12 polymorphic loci are detected directly in the same reaction; in addition, the method has low cost and visual result judgment.

Description

technical field [0001] The invention relates to a method for simultaneously detecting 12 site polymorphisms of human mitochondrial cytb gene, which belongs to the field of genetic engineering. Background technique [0002] With the completion of the Human Genome Sequencing Project, human beings have entered the post-genome era. Research in this era focused on genetic differences among individuals. The most common of such differences are single nucleotide polymorphisms (SNPs), DNA sequence polymorphisms caused by variations of a single nucleotide at the genomic level. Such polymorphisms are major determinants of human disease susceptibility. [0003] Cytochrome (Cytochrome, Cyt) is a kind of chromoprotein containing iron porphyrin prosthetic group, which belongs to electron donating body. Cytb gene (Cytochrome b, Cytb) is the region 14747-15887 on the mitochondria, which is a kind of cytochrome. The Cytb gene expresses the corresponding protein across both sides of the me...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 田亚平董矜
Owner GENERAL HOSPITAL OF PLA
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