Warm start high temperature resistant DNA polymerase, and preparation method

A high-temperature, polymerase technology, applied in the field of protein chemistry in biochemistry, can solve the problems of loss of hot-start effect, poor hot-start effect, cumbersome operation, etc., achieve high retention rate of original enzyme activity, improve production efficiency, and reduce costs. Effect

Active Publication Date: 2010-06-30
AGCU SCIENTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the manual hot-start method is currently less used due to cumbersome operations; the wax protective layer coating method is not an ideal industrialization method due to the complicated process and poor hot-start effect; the antibody method uses antibodies to specifically block the enzyme active center. , but the combination of enzyme and antibody in this method is a non-chemical bond combination, and the risk of antibody activity decline and hot-start effect loss is prone to occur during the storage of the hot-start enzyme. It is not the best hot-start modification method to block the enzymatic activity of high-temperature-resistant DNA polymerase in the true sense; the chemical modification method forms a stable amido bond with the amino group on the peptide chain of high-temperature-resistant DNA polymerase through chemical substances, which can be used at room temperature. Completely block the activity of the enzyme, hydrolyze the amide bond through high temperature, and redisplay the high temperature resistant DNA polymerase activity, so this method is currently one of the most ideal methods for producing hot start high temperature resistant DNA polymerase

Method used

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  • Warm start high temperature resistant DNA polymerase, and preparation method
  • Warm start high temperature resistant DNA polymerase, and preparation method
  • Warm start high temperature resistant DNA polymerase, and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] 1. 2-methyl-2-butene-2-acid succinic anhydride or 2-methyl-2-butene-2-acid-2,3,4,5,6 pentafluorophenolic anhydride modified high temperature resistant DNA Polymerase (Taq enzyme) protein produces a hot start high temperature resistant DNA polymerase. The specific production steps are as follows:

[0025] The structural formula of 2-methyl-2-butene-2-acid succinic anhydride is as follows:

[0026]

[0027] 2-Methyl-2-butene-2-acid succinic anhydride

[0028] The structural formula of 2-methyl-2-butene-2-acid-2,3,4,5,6 pentafluorophenolic anhydride is as follows:

[0029]

[0030] 2-Methyl-2-butene-2-acid 2,3,4,5,6-pentafluorophenol anhydride

[0031] 2. Take the high-temperature-resistant DNA polymerase (Taq enzyme) from Promega Company, the enzyme protein concentration is 50 mg / ml, and the Taq enzyme activity is determined to be 180 U / ul.

[0032] 3. Weigh 50 mg of 2-methyl-2-butene-2-acid succinic anhydride or 2-methyl-2-butene-2-acid-2,3,4,5,6 pentafluorophen...

Embodiment 2

[0037]1. 2,4-Hexadienoic acid succinic anhydride or 2,4-Hexadienoic acid-2,3,4,5,6 pentafluorophenolic anhydride modify high temperature resistant DNA polymerase (Taq enzyme) protein to produce hot start high temperature resistant DNA polymerization enzyme. The specific production steps are as follows:

[0038] The structural formula of 2,4-hexadienoic acid succinic anhydride is as follows:

[0039]

[0040] 2,4-Hexadienoic acid succinic anhydride

[0041] The structural formula of 2,4-hexadienoic acid-2,3,4,5,6 pentafluorophenolic anhydride is as follows:

[0042]

[0043] 2,4-Hexadienoic acid-2,3,4,5,6-pentafluorophenolic anhydride

[0044] 2. Take the high-temperature-resistant DNA polymerase (Taq enzyme) from Promega Company, the enzyme protein concentration is 50 mg / ml, and the Taq enzyme activity is determined to be 180 U / ul.

[0045] 3. Weigh 25 mg of 2,4-hexadienoic acid succinic anhydride or 2,4-hexadienoic acid-2,3,4,5,6 pentafluorophenolic anhydride, and a...

Embodiment 3

[0050] 1. Modify the high temperature resistant DNA polymerase (Taq enzyme) protein with 3,5-dinitrosalicylic acid succinic anhydride or 3,5-dinitrosalicylic acid p-nitrophenolic anhydride to produce hot start high temperature resistant DNA polymerase. The specific production steps are as follows:

[0051] The structural formula of 3,5-dinitrosalicylic acid succinic anhydride is as follows:

[0052]

[0053] 3,5-Dinitrosalicylic acid succinic anhydride

[0054] The structural formula of 3,5--dinitrosalicylic acid p-nitrophenol anhydride is as follows:

[0055]

[0056] 3,5-Dinitrosalicylic acid p-nitrophenol anhydride

[0057] 2. Take the high-temperature-resistant DNA polymerase (Taq enzyme) from Takara Company, the enzyme protein concentration is 30 mg / ml, and the Taq enzyme activity is determined to be 100 U / ul.

[0058] 3. Weigh 1 mg of 3,5-dinitrosalicylic acid succinic anhydride or 3,5-dinitrosalicylic acid p-nitrophenol anhydride, and add it to 50ul of dimethy...

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Abstract

This invention relates to a method for preparing host-starting heat-resistant DNA polymerase. The activity of the host-starting heat-resistant DNA polymerase is reversibly blocked through chemical modification, and can be recovered after treatment at 94 deg.C or higher and pH = 8-9. The host-starting heat-resistant DNA polymerase has low 3'-5' elongation activity at low temperatures, and has suchadvantages as high specificity, high reliability, high uniformity and high sensitivity. The host-starting heat-resistant DNA polymerase can be used in PCR, especially human STR-PCR DNA composite proliferation gene separation.

Description

technical field [0001] The invention relates to a hot-start high-temperature resistant DNA polymerase and a preparation method thereof, more specifically to a hot-start high-temperature resistant DNA polymerase produced by chemical modification and a preparation method thereof, belonging to the field of protein chemistry in biochemistry. Background technique [0002] At present, high-temperature resistant DNA polymerase has been widely used in molecular biology such as gene detection, gene cloning and other technical fields, and its main function is to amplify the signal of the DNA to be detected through DNA polymerase chain reaction. The basic principle is similar to DNA's natural replication process. DNA polymerase chain reaction consists of three basic reaction steps: denaturation-annealing-extension: ① Denaturation of template DNA: After the template DNA is heated to about 93°C for a certain period of time, the template DNA is double-stranded or amplified by PCR to form ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00C12N15/52
Inventor 郑卫国熊勇华陈光辉涂祖新
Owner AGCU SCIENTECH
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