Gene cloning and site-specific mutagenesis method by utilizing high-fidelity polymerase and UDG

Abstract

The invention discloses a gene cloning and site-specific mutagenesis method by using high-fidelity polymerase and UDG. The gene cloning and site-specific mutagenesis method comprises the following steps: amplifying a target gene and a plasmid vector by using a dU modified primer and dU tolerant high-faithfulness DNA polymerase; carrying out high-temperature treatment by using heat-stable uracil DNA glycosidase to obtain a target gene and a plasmid vector of which the 3'end is a single chain; mixing the target gene and the plasmid vector so that the target gene and single-stranded regions at the two ends of the plasmid vector are paired and connected to form a notched annular recombinant plasmid vector containing the target gene; after escherichia coli is transformed by the annular recombinant plasmid vector with the notch containing the target gene, repairing the notch, and obtaining the complete recombinant plasmid vector containing the target gene . By adopting the method, the faithfulness of an amplification reaction is remarkably improved, extra gene mutation caused by Taq DNA polymerase with low faithfulness is avoided, and the method is particularly suitable for vector construction with protein expression as a target; dU and dA bases are paired, the pairing result is equal to normal dT / dA pairing, and gene mutation caused by introduction of additional wrong bases into modified bases is avoided; the thermostable UDG has enzymatic activity at 60-80 DEG C, so that removal of dU bases and breakage of DNA chains are performed simultaneously, and alkali-adding thermal cracking operation is omitted.

Description

technical field [0001] The invention relates to the field of genetic engineering, especially the construction of high-throughput expression vectors, gene site-directed mutation, etc., more specifically, it relates to a gene cloning and gene site-directed mutation method using high-fidelity DNA polymerase and UDG. Background technique [0002] DNA ligases and restriction enzymes make it possible to reconstitute specific DNA in vitro. Reconstructing DNA in vitro, also known as gene cloning or genetic engineering, has greatly promoted the development of modern protein engineering. The traditional gene cloning technology first uses restriction endonuclease to digest the DNA fragment of the target gene and the plasmid vector, and then DNA ligase connects and circularizes the target gene and the plasmid to form a complete recombinant plasmid. Since restriction endonucleases recognize specific DNA sequences, the recognition sequences of different restriction endonucleases are diff...

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Problems solved by technology

However, most high fidelity DNA polymerase activity is inhibited by the dU base

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