Detection of mispairing ratio of DNA polyase in PCR by denatured gradient gel electrophoresis

Abstract

The method incldues following steps. (1) The specific strain is selected to be as the strain used in experiments. (2) After purifying and separating the strains, the selected single colonly liquid is used to cultivate the thallus. (3) The purified gene group DNA of the distilled specific strain is utilized as the template of PCR reaction. (4) After selecting the specificity primer, using different DNa polymerase to be tested carries out the PCR amplification. (5) The denaturation gradient gel electrophoresist separates the PCR products. (6) With the electrophoresis being finished, dyeing bands and counting the number of the each band of specimens obtains the mismatching rate for different DNA polymerases. The invention provides the means for testing the mismatching rate so as to assure the accuracy of the experiments.

Description

Technical field: [0001] Polymerase chain reaction (PCR) technology is a basic technology in the field of molecular biology at present, and it is mainly used to amplify a DNA segment located between two known sequences. It has a wide range of applications in genetics, forensic science and other fields. In addition, PCR amplification technology has the following applications in molecular cloning and DNA analysis: (1) nucleic acid probe production, (2) DNA library establishment, (3) DNA sequence determination (4) mutation analysis, etc. Background technique: [0002] Most of the currently used PCR methods use Taq DNA polymerase purified from Thermus aquaiticus, which has relatively large limitations, mainly manifested in a high rate of incorporation errors. According to statistics, the incorporation error rate of Taq DNA polymerase in the PCR process can reach 2×10 -4 nucleotides, for a 30-cycle amplification reaction, this incorporation error rate will lead to a total error f...

AI Technical Summary

Problems solved by technology

Although everyone has recognized that Pfu DNA polymerase has better fidelity than traditional Taq DNA polymerase in the PCR process, but the specific fidelity rate of DNA polymerase in the PCR process (or vice versa, the mismatch rate) The detection method has not been reported yet. Based on this aspect, this study proposes a detection method, that is, the detection method using denaturing gradient gel electrophoresis (DGGE)

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