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.