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Efficient extraction method for whole blood genome DNA (deoxyribonucleic acid)

An extraction method and genome technology, applied in the field of nucleic acid purification, can solve the problems of expensive instruments and reagents, complicated operation and long time, and achieve the effects of low cost, high purity and good integrity

Inactive Publication Date: 2014-02-12
NORTH CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, one of the difficulties in extracting DNA from blood is that DNA is easily contaminated by various substances, which reduces the purity of DNA and reduces the accuracy of experimental results.
Some traditional peripheral blood DNA extraction methods use harmful substances such as saturated phenol, and the operation is complicated, the process is cumbersome, and the time is too long; although the open automatic DNA extraction device does not use saturated phenol and other harmful substances, the operation process is relatively simple. However, the instruments and reagents used are extremely expensive and are not suitable for large-scale DNA extraction in ordinary laboratories

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Extraction of DNA from human whole blood.

[0036] (1) Add 600 microliters of red blood cell lysate to 600 microliters of human whole blood, the volume ratio added is whole blood: red blood cell lysate = 1:1, vortex for 2 minutes, and stand for more than 5 minutes until the solution is clear and transparent . The solution was centrifuged at a speed of 4000 rpm for 2 minutes, and the supernatant was discarded. Wherein the erythrocyte lysate is prepared as follows: get 10ml1MTris-HCl (PH7.6), 109.54g Sucrose, 1.01g MgCl 2 , add 10ml Triton×100, add water to 800ml, dissolve to 1000ml.

[0037] (2) Add 200 microliters of red blood cell lysate to the EP tube in step (1), and vortex for 1 minute until the precipitate is completely dispersed. The solution was centrifuged at a speed of 4000 rpm for 1 minute, and the supernatant was discarded. Repeat once, and blot the remaining liquid with absorbent paper.

[0038] (3) Add 200 microliters of white blood cell dil...

Embodiment 2

[0045] Example 2: Extraction of DNA from whole blood of mice.

[0046] (1) Add 600 microliters of erythrocyte lysate to 600 microliters of mouse whole blood at a volume ratio of whole blood: red blood cell lysate = 1:1, vortex for 2 minutes, and let stand for more than 5 minutes until the solution is clear transparent. The solution was centrifuged at a speed of 4000 rpm for 2 minutes, and the supernatant was discarded. Wherein the erythrocyte lysate, its preparation method is: get 10ml1MTris-HCl (PH7.6), 109.54g Sucrose, 1.01g MgCl 2 , add 10ml Triton×100, add water to 800ml, dissolve to 1000ml.

[0047](2) Add 200 microliters of red blood cell lysate to the EP tube in step (1), and vortex for 1 minute until the precipitate is completely dispersed. The solution was centrifuged at a speed of 4000 rpm for 1 minute, and the supernatant was discarded. Repeat once, and blot the remaining liquid with absorbent paper.

[0048] (3) Add 200 microliters of white blood cell dilution...

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Abstract

The invention relates to a method for quickly and efficiently extracting whole blood genome DNA (deoxyribonucleic acid), and belongs to the field of nucleic acid purification. The method comprises the following steps: adding a red cell lysis solution into whole blood, eddying until red cells are completely split, removing supernatant, and sucking all residual liquid; adding a white cell lysis solution and protease K, and splitting white cells in a water bath of 37 DEG C until the white cells are deposited and dissolved; adding sodium chloride and chloroform, carrying out centrifugal separation on DNA, and transferring the supernatant to another clean EP (epoxy) pipe; adding ammonium acetate and absolute ethanol for depositing DNA, washing by 75% of ethanol, naturally drying, and adding TE (Tris-EDTA(ethylene diamine tetraacetic acid)) for dissolving to obtain DNA. The method has the characteristics of high speed, high efficiency and non-toxicity. The extracted DNA can be used for various subsequent molecular biology studies such as polymerase chain reaction (PCR), methylation detection and gene cloning.

Description

[0001] technical field [0002] The invention relates to a fast and efficient whole blood genome DNA extraction method, which belongs to the field of nucleic acid purification. Background technique [0003] At present, molecular biology theory and technology are developing very rapidly, and have been closely integrated with biology, medicine, genetics, zoology and other disciplines. Obtaining high-purity and complete genomic DNA is the basis for subsequent molecular biology research. Prerequisites for other research work. Peripheral blood can be used as molecular markers in the research of hematopoietic system diseases, as well as in the development of in vitro diagnostic systems, and can also be used for molecular monitoring of many systemic diseases outside the blood system. Extracting DNA from peripheral blood and conducting research at the nucleic acid level is an important research method in clinical molecular biology. [0004] In the study of genetic diseases, tumors...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 张雪梅张志曹蕾
Owner NORTH CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY
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