Method for rapidly extracting mitochondrial DNA and application and related kit thereof

A technology of mitochondria and kits, which is applied in the direction of DNA preparation, recombinant DNA technology, DNA / RNA fragments, etc., can solve the problems of cumbersome process and many extraction steps of mitochondrial DNA, so as to avoid mutual pollution, achieve consistent results, and reduce work intensity effect

Inactive Publication Date: 2016-07-20
BEIJING BOYI TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In order to solve the technical problem of many extraction steps and complicated process in the prior art, one of the objects of the pres

Method used

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  • Method for rapidly extracting mitochondrial DNA and application and related kit thereof
  • Method for rapidly extracting mitochondrial DNA and application and related kit thereof
  • Method for rapidly extracting mitochondrial DNA and application and related kit thereof

Examples

Experimental program
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Embodiment 1

[0089] This embodiment is an improved method for extracting mitochondrial DNA, the general process is as follows figure 1 As shown, the mitochondrial DNA was obtained by removing red blood cells, lysing white blood cells, and releasing mitochondrial DNA from whole blood, followed by PCR amplification and identification.

[0090] (1) Sample pretreatment (taking clinical whole blood samples as an example)

[0091] (a) Mix 1ml of blood sample with 3ml of erythrocyte lysate, then turn it upside down 6-8 times, let it stand at room temperature for 10 minutes, during this period turn it upside down, then centrifuge at 4000rpm for 5 minutes, discard the supernatant;

[0092] (b) Vortex the leukocyte pellet in the centrifuge tube for 15 seconds to disperse it into single cells;

[0093] (c) Add 1 mL of erythrocyte lysate to the centrifuge tube, then invert it up and down 6-8 times, let it stand for 10 minutes, during this period, centrifuge at 4000rpm for 5 minutes, and discard the s...

Embodiment 2

[0106] Carry out the experiment in the same manner as in Example 1, only replace step (d) and the 10-fold diluted lysis buffer (10×) in Table 1 with 200mM Tris-HCl+1%SDS, the obtained results are as follows figure 2 Shown in f.

Embodiment 3

[0121] (1) Red blood cell removal

[0122] (a) Mix 1ml of blood sample with 3ml of erythrocyte lysate, then turn it upside down 6-8 times, let it stand at room temperature for 10 minutes, during this period turn it upside down, then centrifuge at 4000rpm for 5 minutes, discard the supernatant;

[0123] (b) Vortex the leukocyte pellet in the centrifuge tube for 15 seconds to disperse it into single cells;

[0124] (c) repeat steps (a) (b) once;

[0125] (2) Cell Lysis and Mitochondrial DNA Acquisition

[0126] (d) Add 1 mL of erythrocyte lysate to the centrifuge tube, then invert it up and down 6-8 times, let it stand for 10 minutes, during this period, centrifuge at 4000rpm for 5 minutes, and discard the supernatant;

[0127] (e) blow the white blood cell pellet with a gun to disperse it into single cells, take 70 μl, add 7 μl of lysis buffer (10×) diluted 10 times; the lysis buffer (10×) is 200mMTris-HCl+1%TritonX Aqueous solution of -100;

[0128] (f) Cook the mixture at...

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Abstract

The invention relates to a method for rapidly extracting mitochondrial DNA and application and a related kit thereof. The method comprises the steps that a tissue sample or a blood sample with no red blood cell is dispersed; a lysis buffer is added into the dispersed cell sample, the lysis buffer is a water solution containing Tris-HCl and TritonX-100, the molar concentration of Tris-HCl is 19-21 mM, and the pH of the water solution is 8.7-8.9; the volume concentration of TritonX-100 is 0.09-0.11%; or, the lysis buffer is a water solution containing Tris-HCl and SDS, the molar concentration of Tris-HCl is 19-21 mM, and the pH of the water solution is 8.7-8.9; the mass concentration of the SDS is 0.09-0.11%; a supernate is separated out, and the mitochondrial DNA of the sample is obtained through extraction. By adopting the method, the mitochondrial DNA capable of being adopted as an amplification template can be obtained in a short period of time.

Description

technical field [0001] The invention relates to a method for rapidly extracting DNA and its application, in particular to a method for rapidly extracting mitochondrial DNA, its application and related kits. Background technique [0002] Mitochondrial DNA is the only extranuclear genetic material in human cells. The mitochondrial chromosomes are about 16,000 nucleotides long. The chromosomes are circular, and the mutation rate is higher than that of DNA in the nucleus. Clonic epilepsy, diabetes-deaf syndrome, etc. are related to mitochondrial gene mutations. [0003] Leber hereditary optic neuropathy is a maternally inherited disease of optic nerve degeneration. Most of the patients are male, and the onset usually occurs between 15 and 35 years old. The main clinical manifestations are acute or subacute painless vision loss in both eyes at the same time or successively, accompanied by loss of central visual field and color vision disturbance. The severity of visual impairme...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68C12N15/11
CPCC12Q1/6806C12Q1/6883C12Q2600/156C12Q2531/113C12Q2537/143
Inventor 艾南平魏彦刚
Owner BEIJING BOYI TECH CO LTD
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