A new method for multi-site-directed mutagenesis.
A modified method for multi-site-directed mutagenesis was developed here based on polymerase chain reaction (PCR), DpnI digestion, and overlap extension. It needs only methylated plasmids obtained by Dam methyltransferase or plasmids from dam(+)Escherichia coli containing target gene. The procedure consists of PCR, DpnI digestion, overlap extension PCR, and plasmid transformation. The method was developed for multi-site-directed mutagenesis, including close proximity of mutation sites. It does not require 5'-phosphorylated primers and ligation and, thus, significantly simplifies the routine work and reduces the experimental cost for multi-site-directed mutagenesis.
Tian
Jing
J
Shenzhen Uinversity, People's Republic of China.
Liu
Qiong
Q
Dong
Sheng
S
Qiao
Xifeng
X
Ni
Jiazuan
J
D001483:Base Sequence; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D051140:Selenoproteins / Q000235:genetics
D051140:Selenoproteins; C020102:endodeoxyribonuclease DpnI; D015252:Deoxyribonucleases, Type II Site-Specific