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Chaotropic agent and method for extracting genomic DNA by using chaotropic agent

A chaotropic agent and genome technology, applied in the field of chaotropic agent for rapid separation of histones and genomic DNA, and DNA extraction, it can solve the problems of poor experimental repeatability and stability, high chlorine valence, and strong oxidative properties of reagents. Avoid prolonged digestion, high purity, and high DNA yield effects

Active Publication Date: 2017-08-18
陈礼平 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of this method are: (1) The incubation temperature of proteinase K is 50 to 65°C, requiring additional heating equipment
(2) It takes time for proteinase K to digest histones, usually 0.5h-overnight, and the experiment takes a long time
(3) With the extension of storage time and the increase of repeated freeze-thaw times, the activity of proteinase K will decrease, and the repeatability and stability of the experiment will be poor.
Among them, the reagent concentrations used in the guanidine hydrochloride and guanidine thiocyanate methods are high, although the reagent concentrations used in the sodium perchlorate and lithium perchlorate methods are low, but the valence of chlorine in perchlorate is high, which is +7. Strong oxidizing

Method used

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  • Chaotropic agent and method for extracting genomic DNA by using chaotropic agent
  • Chaotropic agent and method for extracting genomic DNA by using chaotropic agent
  • Chaotropic agent and method for extracting genomic DNA by using chaotropic agent

Examples

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Embodiment approach

[0029] Wherein, as a preferred embodiment of the present invention, the sample containing histone and genomic DNA contains cell lysate.

[0030] Wherein, the method may further include: pretreating the eukaryotic biological material to obtain biological material containing histone and genomic DNA suitable for lysing directly. The pretreatment operation includes at least one of washing, drying, shredding, grinding, freezing and freeze-thawing. The pretreatment operation can be performed in a conventional manner in the art, for example, according to the contents recorded in the "Molecular Cloning Experiment Guide".

[0031] Wherein, the eukaryotic biological material may include at least one of tissues, organs, individuals and symbionts. The biological material may include at least one of oral mucosal epithelial cells, blood and artificially cultured cells.

[0032] Wherein, the method further includes: precipitating, washing and redissolving the DNA. Wherein, the reagent use...

Embodiment 1

[0035] This example is used to illustrate the operation of extracting DNA from saliva samples using two different methods.

[0036] (1) Sample pretreatment: centrifuge saliva at 2000 g for 10 min, discard the supernatant, suspend oral mucosal epithelial cells with 1 mL of normal saline, centrifuge at 2000 g for 10 min, and discard the supernatant.

[0037] (2) Add 400 μL of cell lysate to the Eppendorf (EP) tube, and mix thoroughly until there is no obvious cell mass. The cell lysate formula is: 10 mmol / L Tris-HCl (pH8.0), 30 mmol / L EDTA (pH8.0), 0.5% SDS, RNase A (20 μg / mL).

[0038] (3) Genomic DNA was extracted by proteinase K method and sodium bromate method respectively. Proteinase K method: Add proteinase K with a final concentration of 100 μg / mL to the EP tube in step (2), mix well, and place at 56 °C for 40 min, mixing from time to time. Sodium bromate method: Add chaotropic agent sodium bromate with a final concentration of 1 mol / L to the EP tube in step (2), and mi...

Embodiment 2

[0044] This example is used to illustrate the operation of extracting DNA from oral mucosal epithelial cells collected by oral swab method using two different methods.

[0045] (1) Sample pretreatment: Cut out the material carrying oral mucosal epithelial cells and place it in an Eppendorf (EP) tube.

[0046] (2) Add 400 μL of cell lysate to the EP tube, and mix well until there is no obvious cell mass. The cell lysate formula is: 10 mmol / L Tris-HCl (pH8.0), 30 mmol / L EDTA (pH8.0), 0.5% SDS, RNaseA (20 μg / mL).

[0047] (3) Genomic DNA was extracted by proteinase K method and sodium bromate method respectively. Proteinase K method: Add proteinase K with a final concentration of 100 μg / mL to the EP tube in step (2), mix well, and place at 56 °C for 40 min, mixing from time to time. Sodium bromate method: Add chaotropic agent sodium bromate with a final concentration of 1 mol / L to the EP tube in step (2), and mix well. The rest of the steps are the same.

[0048] (4) Add an e...

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Abstract

The invention discloses a chaotropic agent and a method for extracting genomic DNA by using the chaotropic agent. The chaotropic agent contains sodium bromate, and the compound can rapidly and effectively separate histone in a nucleosomal structure from the genomic DNA. The method for extracting genomic DNA by using the chaotropic agent includes the steps that a cell lysis solution is added in a sample to make cells rupture; the mixture is then mixed evenly with the chaotropic agent, and an object is obtained after the histone and the genomic DNA are separated; protein extracting liquid is added, uniform mixing is conducted, and after centrifugation, supernatant is obtained after protein is extracted; the supernatant is mixed with a DNA precipitant, and after centrifugation, a DNA precipitate is obtained. The method has the following advantages that compared with the method for extracting genomic DNA by using sodium perchlorate, the type of the chaotropic agent is different, the valence is lower, and the yield and purity of the genomic DNA are more appropriate; compared with the traditional protease K method, long-time digestion is not needed, a great deal of time is saved, the defects that the activity of protease K is easy to change and digestion is insufficient are avoided, the repeatability is good and the stability is high.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a chaotropic agent for rapidly separating histone and genomic DNA, and a method for extracting DNA. Background technique [0002] For eukaryotic organisms including humans, the genomic DNA of an organism refers to all the genetic information contained in the DNA of the organism. Genomic DNA and histones are bound together by non-covalent bonds. Therefore, how to separate DNA and histones is the key and difficult point in the process of genomic DNA extraction. In fact, for different genomic DNA extraction methods, the steps of sample pretreatment (cell lysis) in the early stage and DNA precipitation and washing in the later stage are similar. The core difference between different methods lies in how to effectively separate DNA and histones. [0003] The proteinase K method is the most classic method, which uses proteinase K to degrade histones, thereby releasing genomic DNA. The dis...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 陈礼平王海英
Owner 陈礼平
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