Hot start Taq DNA polymerase preparation method

A polymerase and hot-start technology, applied in the biological field, can solve the problems of cumbersome operation steps, loss of activity, and peptide bond breakage in the wax isolation method, so as to avoid non-specific amplification and primer-dimer generation, and improve reaction specificity Effects on Sex and Sensitivity

Inactive Publication Date: 2017-01-25
周辉
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Problems solved by technology

However, the chemical modification and heparin salt method need preheating, which is easy to cause DNA damage; the wax isolation method is cumbersome, and it is eas...

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  • Hot start Taq DNA polymerase preparation method

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[0047] Example: Preparation of Hot Start Taq DNA Polymerase

[0048] 1) Coating and blocking of ELISA plate: 100 μl of Taq enzyme was prepared and coated in well A of a 96-well ELISA plate as the first round of screening solution, and then the concentration of the screening solution was gradually decreased. Then add 100 μl coating solution (0.05mol / L NaHCO3, pH9.6) and mix well. At the same time, set a blank control well—well B, and add 100 μl of deionized water instead of Taq enzyme into well B for coating. Store in a ziplock bag overnight at 4°C. The coating solution was discarded, and the plate was washed 4 times with SELEX washing buffer (SHCMK+0.05% Tween 20), 3 min each time. Wells A and B were treated with 200 μl 3% BSA, blocked at 37°C for 2 hours, and the blocking solution was discarded.

[0049] 2) Binding of ssDNA: Add 100ul of SELEX binding buffer (SHCMK solution: 20mmol / L Hepes pH7.35, 120mmol / L NaCl, 5mmol / L KCl, 1mmol / LCaCl 2 ,1mmol / L MgCl 2 ), then add a c...

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Abstract

The invention relates to hot start Taq DNA polymerase preparation method. The method includes: screening a positive clone bacterium from an artificial synthesis ssDNA library by means of SELEX (systematic evolution of ligands by exponential enrichment), wherein the positive clone bacterium is high in Taq DNA polymerase compatibility and stable in compatibility at a temperature of 45 DEG C or below; further analyzing a sequence of the clone bacterium; artificially synthesizing an ssDNA aptamer, and proportionally dissolving the aptamer and the Taq DNA polymerase in Enhancer solution. The hot start Taq DNA polymerase preparation method has advantages that in a PCR (polymerase chain reaction) process, hot start is realized at the first step as well as follow-up steps of PCR reaction, and accordingly non-specific amplification and primer dimers are avoided, and reaction specificity and sensitivity are improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing a hot-start Taq DNA polymerase. Background technique [0002] Polymerase chain reaction, or PCR technology, is used to amplify specific DNA fragments in vitro without relying on organisms such as Escherichia coli, and is widely used in medical and biological laboratories. The DNA polymerase required for the PCR reaction is currently widely used as a thermostable DNA polymerase produced by the thermophilic bacterium Thermus aquaticus, namely Taq DNA polymerase. However, Taq DNA polymerase lacks 3' to 5' proofreading exonuclease activity, which is prone to non-specific amplification and primer-dimers. In order to solve this technical problem, people invented the hot-start PCR technology to solve it. In the hot-start PCR technology, the addition of a specific hot-start Taq DNA polymerase is required to realize the entire hot-start process. Because the hot-start ...

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Application Information

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IPC IPC(8): C12N9/12C12N15/10
CPCC12N9/1252C12Q1/686C12Y207/07007C12Q2521/101C12Q2541/101
Inventor 周辉
Owner 周辉
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