[A simple error-prone PCR method through dATP reduction].
The error-prone PCR is one of the main methods for in vitro gene mutagenesis, usually through adding Mn2+ increasing Mg2+ and dCTP/dTTP concentration.


OBJECTIVE
In this study, both the antifungal protein gene Ace-AMP1 from Allium cepa and the Bt toxin gene cry1A(c) from Bacillus thuringiensis were subjected to PCR mutagenesis through reducing the dATP concentration, but without adding Mn2+ or adjusting other PCR components.


RESULTS
The result showed that the rates of base mutation and sequence variation were increased along with the decrease of dATP concentrations. When dTTP/dCTP/dGTP: dATP equaled 20:1-40:1, the rate of base mutation was between 1.4% and 1.8%, and the rate of sequence variation was between 77.8% and 100%.


CONCLUSIONS
This method is simple and practical, and enables the process optimization of several mutagenic factors in conventional error-prone PCR. Moreover, as the resulting base mutations were mainly A x T-->G x C transition, the present method provides a new way to improve the GC content of gene by in vitro mutagenesis. The mutagenesis method of simply reducing single dNTP concentration could improve AT or GC content of the target gene, it is an expansion of error-prone PCR.
Yiping
Y
Gao
He
H
Zhao
Mengyu
M
Lv
Guozhong
G
Sun
Xueju
X
Yang
Haibo
H
Wang
D016477:Artifacts; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics; D003838:Deoxyadenine Nucleotides / Q000378:metabolism*; D004731:Endotoxins / Q000235:genetics; D006460:Hemolysin Proteins / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*
C096657:AMP1 protein, Allium cepa; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D003838:Deoxyadenine Nucleotides; D004731:Endotoxins; D006460:Hemolysin Proteins; D010940:Plant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; C026600:2'-deoxyadenosine triphosphate