Method for rapidly extracting DNA from whole blood

An extraction method and technology for blood samples, applied in the field of rapid extraction of DNA from a large number of whole blood, can solve the problems of incompatibility with third-generation sequencing, cumbersome DNA process, long time consumption, etc., and achieve reduction of cleaning and centrifugation steps, simple processing technology, and short time consumption. Effect

Active Publication Date: 2017-08-18
武汉希望组生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In view of this, in order to solve the existing problems of cumbersome process, high cost and long time-consuming, unsuitable for third-generation sequencing, the present invention adopts high-speed centrifugation of a large amount of whole blood to obtain blood cells, and then uses specially prepared whole blood to obtain blood cells. Cell lysate lyses blood cells in one step, and obtains a method suitable for three-generation sequencing to quickly extract a large amount of genomic DNA from blood. This method can obtain qualified DNA within 3.5 hours at the fastest

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  • Method for rapidly extracting DNA from whole blood
  • Method for rapidly extracting DNA from whole blood
  • Method for rapidly extracting DNA from whole blood

Examples

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Embodiment 1

[0039] The present embodiment provides a method for rapid extraction of DNA from whole blood, and the specific steps include:

[0040] 1) Take 7.5ml of human blood sample and thaw it completely at room temperature;

[0041] 2) Centrifuge at 15000rpm at 4°C for 15min, remove the supernatant completely, leaving only the sediment at the bottom of the tube;

[0042] 3) Add 2V of 37°C preheated lysate (0.8M guanidine HCl, 30mM Tris-Cl (pH8.0), 30mM EDTA (pH8.0), 5% Tween-20, 0.5% Triton-100, 0.5% SDS, 0.5mg / ml Proteinse K), use a pipette to thoroughly break up the precipitate and mix it to a clear state;

[0043] 4) Place in a water bath at 56°C for 1 hour, and gently invert and mix once every 10 minutes;

[0044] 5) After cooling to room temperature, add an equal volume of chloroform isoamyl alcohol (24:1) for extraction once;

[0045] 6) Centrifuge at 13000rpm for 10min (16°C), and take the supernatant;

[0046] 7) Add 0.7V frozen isopropanol and 0.1V NaAc (pH5.2), mix well; ...

Embodiment 2

[0057] Take 6ml of monkey blood sample and put it in a 37°C water bath to thaw completely;

[0058] 2) Centrifuge at 15000rpm at 4°C for 15min, remove the supernatant completely, leaving only the sediment at the bottom of the tube;

[0059] 3) Add 2V of 37°C preheated lysate (0.8M guanidine HCl, 30mM Tris-Cl (pH8.0), 30mM EDTA (pH8.0), 5% Tween-20, 0.5% Triton-100, 0.5% SDS, 0.5mg / ml Proteinse K), use a pipette to thoroughly break up the precipitate and mix it to a clear state;

[0060] 4) Place in a water bath at 56°C for 1 hour, and gently invert and mix once every 10 minutes;

[0061] 5) After cooling to room temperature, add an equal volume of chloroform isoamyl alcohol (24:1) for extraction once;

[0062] 6) Centrifuge at 13000rpm for 10min (16°C), and take the supernatant;

[0063] 7) Add 0.7V frozen isopropanol and 0.1V NaAc (pH5.2), mix well;

[0064] 8) Pick out the flocculent precipitate and wash it 3 times with 75% frozen ethanol;

[0065] 9) Dry, add 300ul TEN...

Embodiment 3

[0075] The present embodiment provides a method for rapid extraction of DNA from whole blood, and the specific steps include:

[0076] 1) Take 5ml of human blood sample and thaw it completely at room temperature;

[0077] 2) Centrifuge at 15000rpm at 4°C for 15min, remove the supernatant completely, leaving only the sediment at the bottom of the tube;

[0078] 3) Add 2.5V preheated lysate (1.2M guanidine HCl, 100mM Tris-Cl (pH8.0), 80mM EDTA (pH8.0), 4% Tween-20, 0.3% Triton-100, 0.3 %SDS, 0.3mg / ml Proteinse K), the precipitate was thoroughly dispersed and mixed to a clear state with a pipette;

[0079] 4) Place in a 50°C water bath for 50 minutes, and gently invert and mix once every 5 minutes;

[0080] 5) After cooling to room temperature, add an equal volume of chloroform isoamyl alcohol (25:1) for extraction once;

[0081] 6) Centrifuge at 16000rpm for 8min (10°C), and take the supernatant;

[0082] 7) Add 1V frozen isopropanol and 0.1V NaAc (pH5.2), mix well;

[0083...

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Abstract

The invention provides a method for rapidly extracting DNA from whole blood. The method comprises the following steps: S1, centrifuging a blood sample at a high speed, removing a supernatant, keeping precipitate, adding a lysate, uniformly mixing till a clear state, keeping for 40-60min in a water bath with the temperature of 50-60 DEG C, cooling, performing suction extraction by using an organic solvent, centrifuging, and taking a supernatant; S2, adding isopropanol and NaAc into the supernatant, uniformly mixing, collecting the precipitate, cleaning the precipitate and airing; S3, adding a TEN buffer solution and ribonuclease into the precipitate, uniformly mixing, incubating for 30min, then adding a proteinase K containing TEN buffer solution, uniformly mixing, performing water bath for 30min, cooling, centrifuging, taking a supernatant, and purifying to obtain the pure DNA. By the method, the sample treating amount is large; most of red blood cells in the supernatant are removed through high-speed centrifugation first and then blood cells (white blood cells) in the precipitation are lysed by using the self-prepared whole cell lysate by one step so as to rapidly obtain a large number of DNAs applicable to three generations of sequencing from blood.

Description

technical field [0001] The invention relates to the field of biotechnology, and relates to a method for extracting animal genome DNA, in particular to a method for rapidly extracting a large amount of whole blood DNA for three-generation sequencing. Background technique [0002] Nucleic acid sequencing technology has now become a powerful assistant in the field of biological research. The application of second-generation high-throughput sequencing technology has achieved many gratifying results, but the technical principle determines that there are many disadvantages in second-generation sequencing, such as sequencing read length, Amplification bias caused by repeated PCR amplification, difficulty covering high GC content and repetitive sequence regions, etc. The emergence of the third-generation high-throughput sequencing technology can perfectly solve these problems. Its sequencing read length is 10-60kb or even longer, and there is no PCR amplification process and no GC b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003C12Q1/6806C12Q2527/125
Inventor 汪德鹏陶江明陈逢张笋郭启明王圆黄萌汤环宇
Owner 武汉希望组生物科技有限公司
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