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Method for detecting heterogeneity of mitochondrial genome A3243G site

A mitochondrial genome and site technology, applied in the field of biomedicine, can solve the problems of complex methods for the heterogeneity of the mitochondrial genome A3243G site, and achieve the effect of detection, high specificity and accurate detection

Active Publication Date: 2018-06-22
北京中科卓明生物医学研究所有限公司
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Problems solved by technology

[0004] The present invention provides a new and simplified method for detecting the heterogeneity of the A3243G site in the mitochondrial genome to at least solve the technical problem of complex methods in the prior art for detecting the heterogeneity of the A3243G site in the mitochondrial genome in different tissues

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  • Method for detecting heterogeneity of mitochondrial genome A3243G site
  • Method for detecting heterogeneity of mitochondrial genome A3243G site
  • Method for detecting heterogeneity of mitochondrial genome A3243G site

Examples

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Embodiment 1

[0033] 1. Extraction of peripheral blood or tissue genomic DNA

[0034] 1. Isolation of Peripheral Blood Leukocytes

[0035] (1) Take 5 mL of cubital venous blood from the patient, anticoagulate with EDTA, and centrifuge at 2500 rpm for 10 min;

[0036] (2) Carefully suck off the upper layer of plasma, add 3 times the volume of red blood cell lysate to the lower layer of blood cell pellet, shake well, and ice-bath for 15 minutes.

[0037] (3) Centrifuge at 2 500 rpm for 10 min, discard the supernatant, and repeat step 2 once.

[0038] (4) Centrifuge at 3 000 rpm for 10 min, discard the supernatant, and obtain white blood cells.

[0039] 2. Extraction of leukocyte or tissue DNA

[0040] (1) Take white blood cells, add 600 μL STE lysis buffer, 10 μL proteinase K and 2 μL RNase A, digest at 55°C for 20 minutes (the extraction of tissue DNA is similar to that of white blood cells, cut it with scissors before digestion);

[0041] (2) After the lysate is cooled to room temperatu...

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Abstract

The invention provides a method for detecting the heterogeneity of mitochondrial genome A3243G site. The method comprises the following steps: (a) preparing a standard substance containing wild type and mutant type plasmids in a mitochondrial genome A3243G site fragment; (b) directly taking non-nucleic acid templates as templates to be detected, using a real-time quantification polymerase chain reaction (PCR) technology to carry out single nucleotide polymorphism (SNP) analytic detection on the A3243G site, and using a minor groove conjugate probe (MGB) probe for marking. According to the method, a conventional operation step of extracting nucleic acid DNAs as templates by using PCR is changed, the non-nucleic acid templates such as white cells, urinary sediment, saliva sediment and hair follicles are directly taken as templates, and a preferable result is obtained, so that the detection time is simplified.

Description

technical field [0001] The invention relates to the field of biomedicine, more specifically, to a method for detecting the heterogeneity of the A3243G site of mitochondrial genome in different human tissues. Background technique [0002] Encephalomyopathy with hyperlactatemia and stroke-like episode syndrome (MELAS) is mainly caused by the mutation of adenine (A) to guanine (G) at position 3243 of the human mitochondrial genome (mt.3243A>G), It is the most common manifestation of human mitochondrial disease. In addition to MELAS, mt.3243A>G site mutations can also cause multiple disease phenotypes such as MIDD or stroke. Studies have shown that mitochondrial genome mutations are characterized by heterogeneity, and diseases caused by mitochondrial mutations have a significant threshold effect. Due to the limitations of objective conditions and economic factors, the screening and diagnosis of MELAS patients are still at the level of clinical observation at this stage, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/686
CPCC12Q1/686C12Q1/6883C12Q2600/156C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 荣恩光郝淑静王瀚博刘晓敏王天泽
Owner 北京中科卓明生物医学研究所有限公司
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