In vitro mutagenesis of Xanthomonas campestris alpha-amylase gene by partially replacing deoxythymidine triphosphate with 5-bromo-2'-deoxyuridine-5'-triphosphate using a PCR technique.
Three mutants of the wild type alpha-amylase gene from Xanthomonas campestris pv. campestris 8004 were obtained using a PCR technique in which deoxythymidine triphosphate (dTTP) was partially replaced by 5-bromo-2'-deoxyuridine-5'-triphosphate (BrdUTP), at an optimal dTTP:BrdUTP ratio of 1000:1. Of thre three mutants that were obtained and which were sequenced, one mutant with 40 times higher activity than the wild type alpha-amylase gene product was obtained by using primary PCR products as a template for a second PCR reaction.
Ma
X D
XD
China-UK HUST-RRes Crop Genetic Engineering and Genomics Joint Laboratory, Huazhong University of Science and Technology, Wuhan 430074, PR China.
Ke
T
T
Li
Y X
YX
Huang
C X
CX
Song
A D
AD
Chen
H G
HG
Wu
Y H
YH
Jia
X C
XC
He
G Y
GY
D003001:Cloning, Molecular; D003856:Deoxyuracil Nucleotides / Q000737:chemistry*; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D013942:Thymine Nucleotides / Q000737:chemistry*; D016959:Xanthomonas campestris / Q000235:genetics*; D000516:alpha-Amylases / Q000235:genetics*
D003856:Deoxyuracil Nucleotides; D013942:Thymine Nucleotides; C032549:bromodeoxyuridine triphosphate; D000516:alpha-Amylases; C024157:thymidine 5'-triphosphate