An Yingfeng, Ji Jianfei, Wu Wenfang, Huang Ribo, Wei Yutuo, Xiu Zhilong
Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, China.
Biotechnol Lett. 2008 Jul;30(7):1227-32. doi: 10.1007/s10529-008-9674-9. Epub 2008 Mar 4.
An efficient method for creating a DNA library is presented in which gene mutagenesis and recombination can be introduced by integrating error-prone PCR with a staggered extension process in one test tube. In this process, less than 15 cycles of error-prone PCR are used to introduce random mutations. After precipitated and washed with ethanol solution, the error-prone PCR product is directly used both as template and primers in the following staggered extension process to introduce DNA recombination. The method was validated by using adenosyl-methionine (AdoMet) synthetase gene, sam1, as a model. The full-length target DNA fragment was available after a single round. After being selected with a competitive inhibitor, ethionine, a mutated gene was obtained that increased AdoMet accumulation in vivo by 56%.
本文介绍了一种创建DNA文库的高效方法，该方法可通过在一个试管中将易错PCR与交错延伸过程相结合来引入基因诱变和重组。在此过程中，使用少于15个循环的易错PCR来引入随机突变。易错PCR产物经乙醇溶液沉淀和洗涤后，直接用作后续交错延伸过程中的模板和引物，以引入DNA重组。该方法以腺苷甲硫氨酸（AdoMet）合成酶基因sam1作为模型进行了验证。一轮操作后即可获得全长目标DNA片段。在用竞争性抑制剂乙硫氨酸进行筛选后，获得了一个突变基因，该基因使体内AdoMet积累增加了56%。
