一种降低dATP用量的简单易错PCR方法

A simple error-prone PCR method through dATP reduction

摘要:

摘要:易错PCR是基因体外诱变的主要方法之一，是通过在PCR 体系中添加Mn2+、提高Mg2+浓度和dCTP/dTTP浓度等措施达到基因诱变的目的。【目的和方法】本研究在不添加Mn2+、不调整其它任何PCR成分的情况下，通过降低dATP浓度的底物不平衡作用，对洋葱抗菌蛋白基因Ace-AMP1和苏云金芽胞杆菌毒蛋白(Bt)基因cry1A(c)进行了PCR诱变。【结果】结果表明:随着dATP浓度的降低，序列变异率和碱基突变率均呈升高趋势。当dTTP/dCTP/dGTP:dATP在20:1－40:1时，碱基突变率介于1.4%－1.8%之间，序列变异 率介于77.8%－100%之间。【讨论和结论】该方法简化了常规易错PCR诱变中的条件优化过程，简单实用。降低dATP浓度的诱变方法主要引起AT→GC的变异，适用于提高靶基因GC含量的体外诱变。本研究降低单一底物浓度的诱变方法可以提高诱变基因的AT或GC含量，是对易错PCR诱变方法的拓展。

关键词:关键词:易错PCR，体外诱变，碱基突变，序列变异，洋葱抗菌蛋白基因，Bt 基因

Abstract:

Abstract: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 and Methods］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%．［Discussion and Conclusion］This method is simple and practical，and enables the process optimization of several mutagenic factors in conventional errorprone PCR．Moreover，as the resulting base mutations were mainly A·T→G·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.

Key words:Keywords: Error-prone PCR，In vitro mutagenesis，Base mutation，Sequence variation，Ace-AMP1，Bt