增强的易错滚环扩增突变通过串联体解析。

Enhanced error-prone RCA mutagenesis by concatemer resolution.

机构信息

Department of Biochemistry and Food Chemistry, University of Turku, Tykistökatu 6A, 6th floor, 20520 Turku, Finland.

出版信息

Plasmid. 2011 Oct;66(1):47-51. doi: 10.1016/j.plasmid.2011.03.004. Epub 2011 Mar 29.

DOI:10.1016/j.plasmid.2011.03.004

Abstract

Error-prone rolling circle amplification (RCA) is a promising alternative to error-prone PCR for random mutagenesis. The main disadvantage of error-prone RCA is the low transformation efficiency of the DNA concatemer produced in the amplification reaction. We improved the method by introducing loxP recombination site of bacteriophage P1 Cre recombinase into the target plasmid and reducing the concatemer by Cre recombinase to plasmid-sized units, increasing the number of transformants 50-fold in non-error-prone and 13-fold in error-prone conditions. The efficiency improvement was verified by obtaining 115 ± 57 ceftazidime resistant colonies per recombined RCA reaction from randomly mutated TEM-1 β-lactamase gene library whereas only 9 ± 11 colonies were gained without recombination. Supplementation of the error-prone RCA with Cre/loxP recombination is a simple and useful tool to increase the transformable library size.

摘要

易错滚环扩增（RCA）是一种有前途的替代易错 PCR 的随机诱变方法。易错 RCA 的主要缺点是扩增反应中产生的 DNA 串联体的转化效率低。我们通过在靶质粒中引入噬菌体 P1 Cre 重组酶的 loxP 重组位点，并通过 Cre 重组酶将串联体减少到质粒大小的单位，从而改进了该方法，在非易错条件下将转化体的数量增加了 50 倍，在易错条件下增加了 13 倍。通过从随机突变的 TEM-1 β-内酰胺酶基因文库中每轮重组 RCA 反应获得 115±57 个头孢他啶抗性菌落来验证效率提高，而没有重组则仅获得 9±11 个菌落。易错 RCA 与 Cre/loxP 重组的补充是增加可转化文库大小的简单而有用的工具。