DNA polymerase coding DNA, enzyme coded thereby, and application and preparation method of DNA polymerase

Abstract

The invention belongs to the field of gene engineering, and relates to a DNA polymerase gene separated from Hyperthermophilic Archaeon, a plasmid thereof, a DNA polymerase coded thereby, a preparation method of the DNA polymerase, and an application of the DNA polymerase, and concretely relates to DNA polymerase coding DNA separated from Pyrococcus yayanosii, and an application and a preparation method thereof. The sequence of the DNA polymerase coding DNA is one of a nucleotide sequence represented by SEQ ID NO.1in a sequence table, and a nucleotide sequence obtained by deleting, inserting and substituting one or more nucleotides into the sequence represented by SEQ ID NO.1. The encoded DNA polymerase has the advantages of super strong high-temperature resistance, high fidelity performance, excellent anti-inhibitor ability, and realization of a direct PCR reaction by using crude samples.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to DNA encoding DNA polymerase isolated from hyperthermophilic archaea and the encoded DNA polymerase, application and preparation method thereof, more specifically to the encoding DNA polymerase isolated from Pyrococcusyayanosii DNA and its encoded DNA polymerase, application and preparation method. Background technique [0002] With the development of molecular biology, Polymerase Chain Reaction (PCR) is more and more widely used in gene cloning, diagnosis of genetic diseases and pathogenic microorganisms, individual typing, criminal investigation, archaeology and other fields. Different applications have different requirements for PCR. For example, gene cloning needs to ensure that the target gene can be faithfully amplified; molecular diagnosis needs to ensure the sensitivity of amplification; The templates of similar origin can be amplified smoothly. At present, PCR reactions...

AI Technical Summary

Problems solved by technology

In order to remove inhibitors from templates from different sources, nucleic acids are currently purified through tedious steps such as lysing tissues, organic solvent extraction, and precipitating nucleic acids. The use of organic solvents has a certain toxic effect, and it is time-consuming and labor-intensive. Too many samples are prone to cross-contamination, so Direct nucleic acid amplification using crude samples has great advantages

The direct PCR kit based on Taq DNA polymerase used on the market cannot tolerate high denaturation temperature, cannot effectively release nucleic acid, and cannot amplify well for high GC templates; another high-fidelity enzyme 3 is missing through mutation The '-5' exonuclease activity may be mixed with Taq DNA polymerase in different proportions, so that it can be amplified in whole blood. Due to the loss of correction activity, the error rate of amplification is greatly increased, and it is not good for animal and plant tissues. Ground amplification (PCT patent, WO2009 / 140497)

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