Amplification method, primer, sequencing method and mutation detection method of mitochondria whole genome DNA (Deoxyribonucleic Acid)

A whole-genome sequencing and whole-genome technology, applied in the field of molecular biology, can solve the problems of complicated operation and inability to detect copy number, etc., and achieve the effect of flexible cost and low cost

Inactive Publication Date: 2013-06-26
BGI GENOMICS CO LTD
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Problems solved by technology

Due to the limitation of the length of the sequencing fragment by the fluorescent labeling sequence analyzer, it is necessary to use 26 or more pairs of primers to perform multiple PCR on the mitochondrial genome, and divide it into 26 or more fragments for sequencing. The operation is too cumbersome and cannot be detected. very low copy number variants

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  • Amplification method, primer, sequencing method and mutation detection method of mitochondria whole genome DNA (Deoxyribonucleic Acid)
  • Amplification method, primer, sequencing method and mutation detection method of mitochondria whole genome DNA (Deoxyribonucleic Acid)
  • Amplification method, primer, sequencing method and mutation detection method of mitochondria whole genome DNA (Deoxyribonucleic Acid)

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[0075] See Table 1 for the PCR primers used for mitochondrial whole genome sequencing of the present invention.

[0076] The present invention is based on the method of multiplex PCR amplification with PCR primers for mitochondrial whole genome sequencing, using 8 pairs of PCR primers covering the full length of the mitochondrial genome to amplify the target fragment, then constructing a DNA Paired-End PCR Free Index library, and Miseq sequencing to detect mitochondria The whole genome sequence, the mitochondrial sequence variation information of the sample is obtained through data analysis and comparison.

[0077] PCR amplification

[0078] Eight pairs of PCR primers (Table 1) for mitochondrial genome sequencing were selected, and the upstream and downstream primers were diluted to 10 μmol / L as the working solution. The total volume of the PCR reaction system is 25 μl, which contains 1 μl of DNA template (containing at least 50 ng of whole genome DNA). The DNA template comes...

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Abstract

The invention discloses a primer pair and a kit for amplification of a mitochondria whole genome. The invention further discloses an amplification method, a sequencing method and a mutation detection method of the mitochondria whole genome DNA (Deoxyribonucleic Acid). According to the invention, the direct amplification of the given primer is combined with the direct sequencing method, so that a mitochondria whole genome sequence can be obtained; all mutations of the mitochondria genes can be detected by one step for being used for detecting the diseases caused by most of the mitochondria gene mutations.

Description

technical field [0001] The present invention relates to molecular biology, in particular to a primer pair set, a kit, an amplification method, a sequencing method and a mitochondrial DNA mutation detection method that can be used for the amplification of mitochondrial whole genome DNA. Background technique [0002] The mitochondrial genome (mitochondrial DNA, mtDNA) is a closed double-stranded circular structure with a length of 16569 bp, which is divided into coding regions and non-coding regions. There are a total of 37 genes in the coding region, of which 22 encode transfer ribonucleic acid (tRNA), 2 encode ribosomal ribonucleic acid (12S and 16S rRNA), and 13 encode polypeptides. The mtDNA genes are closely arranged, and there are no introns between the sequences. [0003] The molecular structure of mtDNA determines that it has the following unique genetic characteristics: [0004] (1) High copy number. Generally speaking, there are only two copies of chromosomal DNA i...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/10C12Q1/68
Inventor 费凌娜尉姗姗高景红叶李莉田玉娇易鑫
Owner BGI GENOMICS CO LTD
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