Human mitochondrial DNA (deoxyribonucleic acid) purification method

A purification method, mitochondrial technology, applied in the field of nucleic acid extraction and purification in molecular biology, can solve problems such as cumbersome operating procedures, hinder application, and affect the analysis of trace cell mtDNA, and achieve the effect of simple operation, short time consumption and high purity

Inactive Publication Date: 2012-04-18
WENZHOU MEDICAL UNIV
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  • Abstract
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Problems solved by technology

In China, in order to overcome the shortcomings of traditional methods such as expensive equipment and reagents and cumbersome operating procedures, Hu Yide et al. extracted mtDNA from human peripheral blood leukocytes by improving the alkali denaturation method (publication number: 101338310); Dai Jigang et al. established the Triton method, first Remove the nucleus, and then separate and extract the mtDNA in the cytoplasm (Dai Jigang et al. Journal of Third Military Medical University. Vol.22, No.4: 391-392); Han Dongyi et al improved the high salt method by adding protein denaturant ( Publication number: 101250522)
Although the above methods can obtain a relatively high concentration of mtDNA, there are still certain defects, especially nDNA residues, which hinder the application of related technologies in the field of mtDNA research. For example, there are a large number of mtDNA pseudogenes in nDNA. Failure to remove the interference of these pseudogenes will seriously affect the analysis of trace cell mtDNA

Method used

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  • Human mitochondrial DNA (deoxyribonucleic acid) purification method
  • Human mitochondrial DNA (deoxyribonucleic acid) purification method
  • Human mitochondrial DNA (deoxyribonucleic acid) purification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Effects of different enzyme treatments on whole genome DNA

[0031] Implementation steps:

[0032] 1. Whole Genome DNA Extraction

[0033] (1) Cell line and cell culture: The experiment selected human cervical cancer cell HeLa, cultured it in DMEM culture medium supplemented with 10% fetal bovine serum, placed at 37°C, saturated humidity, containing 5% CO 2 incubator.

[0034] (2) Washing and collection of cells: wash the cultured monolayer with PBS solution, scrape the cells directly, and suspend them with PBS solution to adjust the cell concentration.

[0035] (3) According to the whole genome extraction kit instructions from the cultured HeLa cells (~1.5×10 7 Whole-genome DNA was extracted from cells / dish).

[0036] 2. mtDNA extraction and purification

[0037] (1) Aspirate 4 μg DNA from the whole genome DNA solution, construct 100 μL enzyme digestion reaction system, number 2, which contains 20 μg RNase A, 20U Bgl II, 40U Dra III, 10 μL 10× buffer I an...

Embodiment 2

[0042] Example 2: Changes in concentration and purity of mtDNA before and after purification

[0043] Implementation steps:

[0044] 1. Alkaline lysis method to extract mtDNA

[0045] (1) cultivation and collection of cells (with embodiment 1)

[0046] (2) at every 10 7 Take the number of cells as an example, add 150 μL of solution I, blow and mix in ice bath, add 300 μL of solution II in turn, denature in ice bath for 8 minutes, then add 225 μL of solution III, and refold in ice bath for 25 minutes.

[0047] (3) Centrifuge the lysate at 10,000×g for 6 minutes, take the supernatant, add half volume of Tris saturated phenol (pH>7.6), shake gently for 10 minutes, then add half volume of chloroform / isoamyl alcohol (24:1), shake for 5 minutes , centrifuge at 10,000×g for 10 minutes, take the water phase, add 2 times the volume of absolute ethanol to ice bath for 30 minutes, centrifuge at 15,000×g for 10 minutes, wash with 70% ethanol, centrifuge at 15,000×g for 2 minutes, remov...

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Abstract

The invention relates to a human mitochondrial DNA purification method. The method utilizes the joint action of restriction endonucleases (Bg1 II and Dra III), ribonuclease A and exonuclease III to degrade nuclear DNA and RNA (Ribonucleic acid) residues in genome DNA or mitochondrial DNA crude extract, so that high-purity mitochondrial DNA can be obtained. The method is widely applicable to the purification of human mitochondrial DNA samples, the purity is high, and the method is easy to operate, takes short time, and can meet the requirement of each technology in the mitochondria research field on the high purity of mitochondrial DNA.

Description

technical field [0001] The invention belongs to the technical field of molecular biology nucleic acid extraction and purification, and in particular relates to a human mitochondrial DNA purification method and application thereof. Background technique [0002] Mitochondria, known as the "engine of life", play an important role in the occurrence, development and continuation of life. Mitochondria have relatively independent genetic material. Compared with nuclear DNA (nDNA, nDNA), mitochondrial DNA (mitochondrial DNA, mtDNA) has a different molecular structure and genetic code. Human mtDNA is a covalently closed, circular, double-stranded structure consisting of 16569 base pairs (base pair, bp), encoding 37 genes related to electron transport chain and mitochondrial protein synthesis. Research on disease occurrence, apoptosis and biological evolution around mitochondria has become a hot spot in the field of life sciences. [0003] Obtaining high-purity mtDNA from cells is a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 管敏鑫龚莎莎郑静张婷方芳郑斌娇梁敏吕建新
Owner WENZHOU MEDICAL UNIV
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