Primers, method and kit for whole genome detection of human mitochondria

A whole-genome and mitochondrial technology, applied in the field of gene mutation detection in biotechnology, can solve the problems of low sensitivity, long cycle, high cost, etc., and achieve the effects of improving primer specificity, simple operation, and cost saving

Pending Publication Date: 2020-03-17
杭州联川基因诊断技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to provide a primer, method and kit for detecting mutation sites in the coding sequence of the whole human mitochondrial gene based on multiplex

Method used

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  • Primers, method and kit for whole genome detection of human mitochondria
  • Primers, method and kit for whole genome detection of human mitochondria
  • Primers, method and kit for whole genome detection of human mitochondria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] A series of original templates with different mutation frequencies were used to simulate a series of original templates with known site mutations to verify the performance of this kit.

[0030] A. Template preparation for different mutation ratios

[0031] Plasmid DNA mutated at known sites (chrMT:11778G>A, GRCh37.p13) was mixed with wild-type DNA in proportion to obtain original templates with mutation frequencies of 0.05%, 1%, 2%, 5%, and 10%, respectively .

[0032] B. Multiplex PCR method to amplify the target region of the sample genome

[0033] 1). Prepare a sterile, nuclease-free 200 μL PCR tube and place it on ice; prepare a PCR reaction system as shown in the table below. The amount of template for each reaction is 10ng, and be careful to avoid cross-infection.

[0034] components volume 2×PCR mix 6.25 μL specific primer 0.5μL Universal Primer R5 0.5μL Universal Primer R7 0.5μL DNA template, 50ng 1μL nuclease...

Embodiment 2

[0065] Genomic DNA with different degrees of degradation was used as a template to analyze the correlation between the sequencing results to verify the performance of the kit.

[0066] A. Genomic DNA preparation with different degrees of degradation

[0067] Using 250ng Hela genomic DNA (NEB, N4006S) as raw material, use dsDNAFragmentase (NEB, M0348S) digested the raw materials at 37°C for 0, 10, 20, 30, 50min and other different times, and the digested products were processed by Agilent 2100 instruments for quantitative and quality inspection. Such as figure 1 As shown, the raw material was obviously degraded after being digested at 37°C for 20 minutes, and the longer the treatment time, the more serious the degradation.

[0068] B. Multiplex PCR method to amplify the target region of the sample genome

[0069] 1). Prepare a sterile, nuclease-free 200 μL PCR tube and place it on ice; prepare a PCR reaction system as shown in the table below. The amount of template for ...

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Abstract

The invention discloses primers, method and kit for whole genome detection of human mitochondria. The primers comprise forward and reverse primers for amplifying 37 gene coding regions of a mitochondrial whole genome (16569bp), and the base sequences of the forward and reverse primers are shown in the formulas of SEQ ID NO: 3-222. A detection method based on the NGS technology comprises the following steps: combining specific primers and a target template DNA sequence, amplifying a target region of a to-be-detected sample by using universal primers, purifying a library by using magnetic beads,performing high-throughput sequencing on the obtained library, and analyzing mutation of the mitochondrial gene. According to the primers, the method and the kit disclosed by the invention, the raremutation of mitochondrial DNA can be accurately detected, a high cost performance, least manual operation and ultrahigh sensitivity are realized, guidance for drug selection of familial mitochondrialpatients and genetic susceptibility is provided, mitochondrial diseases caused by mitochondrial DNA mutation assists diagnosis and the onset risk is effectively reduced.

Description

technical field [0001] The invention relates to the field of gene mutation detection of biotechnology, and more specifically relates to a primer, a method and a kit for detecting the whole genome of human mitochondria. Background technique [0002] Mitochondria (mitochondria) is the main site of cellular material oxidation and energy supply center. It can provide more than 90% of adenosine triphosphate (ATP) required for metabolism, regulate apoptosis, and is the main place for intracellular free radicals (ROS) generation. Mitochondria contain DNA molecules, known as human chromosome 25, which is an organelle containing genetic information and expression systems outside the nucleus, and its genetic characteristics are non-Mendelian inheritance, also known as extranuclear inheritance. Mitochondrial disease (mitochondriopathy) is a group of multi-system diseases or tissue-specific diseases caused by defects in mitochondrial metabolic enzymes caused by genetic defects, resulti...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2537/143C12Q2535/122C12Q2545/113
Inventor 潘石玄伟李璐璐王其成林彬钱海峰孙之彬宋金宇
Owner 杭州联川基因诊断技术有限公司
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