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Method for simply and quickly extracting and purifying Taq-DNA polymerase

A polymerase, rapid technology, applied in the fields of biochemistry and molecular biology, can solve the problems of long dialysis time, loss of Taq-DNA polymerase, enzyme inactivation, etc., to simplify the extraction and purification of Taq-DNA polymerase steps, shorten operation time, and save cost

Inactive Publication Date: 2015-01-28
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most laboratories use this method for self-made Taq-DNA polymerase, but we found that the Taq-DNA polymerase produced by this method contains a small amount of DNA, and ammonium sulfate precipitation causes a large loss of Taq-DNA polymerase. Dialysis takes more time
In 2012, Zhong Xing et al. used transgenic Escherichia coli to directly extract the Taq-DNA polymerase secreted into the medium after simple heating and centrifugation, but they did not provide a method for enzyme concentration and purification
We have tried to use this method to isolate Taq holoenzyme, but unfortunately, the boiling water bath cannot effectively lyse Escherichia coli, and the isolated product cannot be stored for a long time, and the enzyme will be inactivated if stored at -20°C for two days

Method used

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  • Method for simply and quickly extracting and purifying Taq-DNA polymerase
  • Method for simply and quickly extracting and purifying Taq-DNA polymerase
  • Method for simply and quickly extracting and purifying Taq-DNA polymerase

Examples

Experimental program
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Effect test

Embodiment 1

[0037] According to the above experimental scheme, Taq-DNA polymerase was extracted. In order to detect the effect of DNase I and RNase A on the DNA and RNA in the enzyme solution, the cells were lysed-heated and centrifuged-the supernatant was taken, and the supernatant was added without adding RNase A and DNase I, DNase I only, RNase A only, and RNase A and DNase I at the same time are variables. After treatment, the sample is reserved, and the rest is precipitated with ethanol. figure 1 It is the result of agarose electrophoresis of 10 μL sample solution. It can be seen from the figure that DNase I and RNase A can effectively remove DNA and RNA in the enzyme solution.

Embodiment 2

[0039] At present, the method of Pluthero FG (1993) is mainly used to prepare Taq-DNA polymerase in the laboratory. For specific operations, refer to the paper Rapid purification of high-activity Taq DNA polymerase. Nucleic acids research, 1993, 21(20): 4850-1. We prepared and purified Taq-DNA polymerase according to its protocol. Electrophoresis detection found that the enzyme solution prepared by this method contained a small amount of DNA contamination, while the enzyme prepared by us had no DNA contamination ( figure 2 ).

Embodiment 3

[0041] In order to identify the effects of different concentrations of ethanol on Taq-DNA polymerase, we tested the purification effect of different concentrations of ethanol on Taq-DNA polymerase. Escherichia coli strains containing Taq-DNA polymerase were lysed, treated with DNase I and RNase A, divided into six equal parts, added ethanol to 40%, 45%, 50%, 55%, 66.7% and 70%, centrifuged Precipitate Taq-DNA polymerase. Then add an equal amount of Storage buffer to dissolve the enzyme, and take an equal amount of enzyme solution to detect its activity. See the test results image 3 . from image 3 It can be seen that the enzymes precipitated with different concentrations of ethanol have little difference in enzyme activity, but the Taq DNA polymerase precipitated with a final concentration of ethanol of 55% has the highest activity. At the same time, take an equal amount of enzyme solution, and detect the purity of the protein in the enzyme solution by polyacrylamide gel ...

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Abstract

The invention provides a method for simply and quickly extracting and purifying Taq-DNA polymerase. According to the method disclosed by the invention, by sufficiently utilizing the precipitation and the nuclease activity of the alcohol on the protein, a step of extracting and purifying the Taq-DNA polymerase is simplified, so that the operation time is shortened and the cost is saved. Compared with the known extracting method, the method disclosed by the invention has the characteristics of simplicity, quickness and low cost, and can be used for completely removing the nucleic acid pollution in the Taq-DNA polymerase extract. The extracted Taq-DNA polymerase can be applied to various PCRs (polymerase chain reactions) which include a real-time quantitative PCR reaction.

Description

technical field [0001] The invention belongs to the fields of biochemistry and molecular biology, relates to a simple and fast method for extracting and purifying Taq-DNA polymerase, which is suitable for preparing Taq-DNA polymerase in mass low-cost commercial production. Background technique [0002] PCR technology is widely used in experiments in biology, agronomy and medicine, and it is isolated from heat-resistant bacteria Thermus aquaticus DNA polymerase (Taq) is a key factor in PCR technology. The gene encoding the enzyme has been transferred into Escherichia coli for high-efficiency expression, which greatly improves the yield of the enzyme and greatly reduces the cost of PCR technology. The Taq-DNA polymerase holoenzyme consists of 832 amino acids, with a size of 94kD; the 5' to 3' exonuclease activity of the enzyme is removed, and the size becomes 61kD, which is called the Stoffel fragment. [0003] Lawyer et al first expressed Taq-DNA polymerase gene in Escheri...

Claims

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Application Information

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IPC IPC(8): C12N9/12
CPCC12N9/1252C12Y207/07007
Inventor 孙新立陈思雀
Owner FUJIAN AGRI & FORESTRY UNIV