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Modified high-salt method for extracting mitochondria DNA and uses thereof

A mitochondrial, high-salt technology, applied in the field of DNA extraction, can solve the problems of short operation time, difficult to repeat, strict requirements, etc., and achieve the effect of low cost and easy operation.

Inactive Publication Date: 2008-08-27
ドングァン アオマイヤ ジェネティック テクノロジー カンパニー リミテッド
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These three methods have their own advantages and disadvantages: the alkali denaturation method has a short operation time, but the requirements are relatively strict, it is not easy to repeat, and is not suitable for clinical mass application; Easy to degrade
The high-salt method is simple to operate, but the commonly used high-salt is mainly sodium chloride, sodium acetate, etc., which only have the effect of precipitating DNA, and the extracted mtDNA has a low purity and is difficult to control.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1: Comparison of different concentrations of guanidine isothiocyanate treatment and traditional high-salt method for extracting mitochondrial DNA:

[0017] Experimental steps:

[0018] (1) Fresh anticoagulated blood or frozen anticoagulated blood

[0019] 1. Take 300μl whole blood. It is best to remove red blood cells first, then add 900μl red blood cell lysate (0.16M NH 4 Cl, 0.13mM EDTA, 12mM NaHCO 3 ; 16.96mmol / L Tris, adjust the pH to 7.2 with 1mol / L HCL, or directly use commercial red blood cell lysate) Gently mix 5 to 6 times, room temperature or ice bath for 10 minutes, 8000rpm centrifuge for 20 to 30 seconds, Discard the supernatant. If the erythrocyte lysis is not complete, the precipitate will turn red, add 900 μl erythrocyte lysate, and repeat the above steps once. If whole blood is frozen before use, repeat steps 1 and 2 twice.

[0020] 2. Add 300 μl of cell cleaning solution to the precipitate, the cell cleaning solution is PBS buffer solution ...

Embodiment 2

[0034] Example 2: Comparison of different concentrations of guanidine hydrochloride treatment and traditional high-salt method for extracting mitochondrial DNA

[0035] Experimental steps:

[0036] 1. Take 300μl whole blood. It is best to remove red blood cells first, then add 900μl red blood cell lysate (0.16M NH 4 Cl, 0.13mM EDTA, 12mM NaHCO 3 ; 16.96mmol / L Tris, adjust the pH to 7.2 with 1mol / L HCL, or directly use commercial red blood cell lysate) Gently mix 5 to 6 times, room temperature or ice bath for 10 minutes, 8000rpm centrifuge for 20 to 30 seconds, Discard the supernatant. If the erythrocyte lysis is not complete, the precipitate will turn red, add 900 μl erythrocyte lysate, and repeat the above steps once. If whole blood is frozen before use, repeat steps 1 and 2 twice.

[0037] 2. Add 300 μl of cell cleaning solution to the precipitate, the cell cleaning solution is PBS buffer solution with pH 7.4, mix well under mild conditions, room temperature or ice bath ...

Embodiment 3

[0051] Embodiment 3: the comparison of using column adsorption centrifugation method and traditional method in combination

[0052] Experimental steps:

[0053] 1. Take 200μl of fresh, frozen or blood with various anticoagulants, and put it into a 1.5ml centrifuge tube. If the initial volume of whole blood is less than 200 μl, make up to 200 μl with buffer or normal saline. If the initial volume is between 200 μl and 300 μl, the subsequent operation needs to increase the reagent volume proportionally. If the initial volume is between 300 μl and 1ml, red blood cell lysis is required first.

[0054] 2. Add 20μl proteinase K (20mg / ml) solution, mix thoroughly, then add 200μl cell lysate containing 5mol / L guanidine isothiocyanate or guanidine hydrochloride, vortex immediately and mix well, place at 70°C for 10 minute. The solution should be clear (but darker in color).

[0055] Optional step, generally not required: If there is a lot of RNA residue and RNA needs to be removed...

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PUM

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Abstract

The invention relates to a method for extracting genome and mitochondria DNA with a high salt method which is modified on the basis of the high salt method and the application thereof. Protein denaturant which contains guanidinium thiocyanate, guanidine hydrochloride and the like is used to crack cells in the method for extracting mitochondria DNA with the modified high salt method, which not only has the effect of depositing protein, but also has the effect of inhibiting nuclease, and the purity and the output of DNA can be greatly increased. The method can be faster and more convenient after being matched with an adsorption column, the operation of a single sample can be normally finished within 20 minutes, the high purity can be guaranteed through rinsing the column for many times, and the method can be directly used in PCR, Southern-blot and various endonuclease reactions. The method has convenient operation and low cost, and is in particular suitable for extracting the genome and the mitochondria DNA from clinical specimens (in particular in peripheral blood).

Description

technical field [0001] The invention relates to a method for extracting DNA, which is a method for extracting chromosomal DNA and organelle DNA from cells, in particular to a method for extracting mitochondrial DNA and its application. Background technique [0002] Mitochondria, an important organelle in cells, play an important role in apoptosis, aging and programmed death. Mutations in the mitochondrial DNA (mtDNA) of patient cells have been found in a variety of diseases. Research on the molecular genetic mechanism of this mitochondrial disease can further clarify the etiology of these diseases and provide theoretical guidance for treatment. It is the most basic work to isolate and extract this mtDNA from cells. Hu Yide, Tamura, and Palva et al. extracted mtDNA from human blood leukocytes and myocardium by alkaline denaturation method; Dai Jigang et al. established the Triton method to first remove the nucleus and then separate and extract the mtDNA in the cytoplasm. T...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12P19/34
Inventor 韩东一戴朴段海清刘丽贤金政策
Owner ドングァン アオマイヤ ジェネティック テクノロジー カンパニー リミテッド
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