Construction of a
high-efficiency multi-site-directed mutagenesis
Yueguang Li2, Ling Chen3, Tomonari
Kasai3 and Masaharu Seno3*
Department, Dalian Institute of Chemical Physics, Chinese
Academy of Sciences, 457 Zhongshan Road, Dalian 116023,
People’s Republic of China.
of General Surgery, Tianjin 4th Centre Hospital, Zhongshanlu,
Hebeiqu, Tianjin 300140, People’s Republic of China.
of Medical and Bioengineering Science, Graduate School of
Natural Science and Technology, Okayama University, Okayama
Polymerase chain reaction; MSD, multi-site-directed
mutagenesis; EK, enterokinase; LB, lysogenic
broth; ccc, circular covalently closed.
Accepted 15 November, 2010
site-directed mutagenesis has been used in many fields, it
still has low rate of success and high cost because of
low-yield target products. A
method for multi-site-directed mutagenesis
with shifted primer design and cold-start polymerase chain
reaction (PCR). The developed method was successfully
applied to hexapeptide gene synthesis and recombinant
enterokinase gene modification in the plasmids pET41a and
pET24b-EK. The efficiency was pronounced at a 1:10 molar
ratio of 7-base mutant products to 705-bp fragment products
as control. Even in a 10-base substitution mutagenic PCR, a
1:50 molar ratio of mutant products to 705-bp fragment
products was reached. Meanwhile, the quality of mutants was
proved through the transformation efficiency and sequencing.
This method was beneficial to prepare high-quality multibase
mutagenesis and also implied that large-scale multibase
mutagenesis was feasible, efficient, economical, and
Site-directed multibase mutagenesis, shift primer,
hexapeptide gene, enterokinase gene.