Enhanced error-prone RCA mutagenesis by concatemer resolution
Enhanced error-prone RCA mutagenesis by concatemer resolution
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DOI：
10.1016/j.plasmid.2011.03.004
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发表时间：
2011-10-01
期刊：
影响因子：
2.6
通讯作者：
Lamminmaki, Urpo
中科院分区：
文献类型：
--
作者：
Huovinena, Tuomas;Julin, Marja;Lamminmaki, Urpo
易错滚环扩增 (RCA) 是用于随机诱变的易错 PCR 的有前途的替代方案。 RCA易错的主要缺点是扩增反应中产生的DNA多联体的转化效率低。我们改进了该方法，将噬菌体 P1 Cre 重组酶的 loxP 重组位点引入目标质粒，并通过 Cre 重组酶将多联体还原为质粒大小的单位，使不易出错条件下的转化体数量增加了 50 倍，易错条件下的转化体数量增加了 13 倍。通过从随机突变的 TEM-1 β-内酰胺酶基因库中每次重组 RCA 反应获得 115±57 个头孢他啶抗性菌落来验证效率的提高，而未经重组仅获得 9+/-11 个菌落。用 Cre/loxP 重组补充容易出错的 RCA 是增加可转化文库大小的简单而有用的工具。 (C) 2011 Elsevier Inc. 保留所有权利。
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 beta-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. (C) 2011 Elsevier Inc. All rights reserved.