Creating random mutagenesis libraries using megaprimer PCR of whole plasmid
Creating random mutagenesis libraries using megaprimer PCR of whole plasmid
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DOI：
10.2144/02335st03
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发表时间：
2002-11-01
期刊：
影响因子：
2.7
通讯作者：
Takenouchi, M
中科院分区：
文献类型：
--
作者：
Miyazaki, K;Takenouchi, M
克隆DNA片段的常规方法是将其插入载体并连接。虽然这种方法很常用，但它是劳动密集型的，因为 DNA 插入片段和载体的比例和浓度需要优化。即便如此，生成的文库通常会受到没有插入片段或多个插入片段的不需要的质粒的困扰。必须消灭这些物种以避免繁琐的筛选，特别是在产生突变基因库时。为了克服这些问题，我们修改了 QuikChange(TM) 方案，使每个质粒都携带单个插入片段。尽管 QuikChange 最初是为在整个质粒 PCR 中使用互补诱变寡核苷酸引物进行定点诱变而开发的，但我们发现该方案也适用于由数百个核苷酸组成的大型引物。基于这一发现，我们使用想要克隆的插入片段作为 QuikChange 反应中的引物。所得文库实际上不含没有插入片段或多个插入片段的物种。因此，我们将本方法命名为 MEGAWHOP（整个质粒的大型引物 PCR），因此非常适合创建随机诱变巨型文库。
The conventional method for cloning a DNA fragment is to insert it into a vector and ligate it. Although this method is commonly used, it is labor intensive because the ratio and concentrations of the DNA insert and the vector need optimizing. Even then, the resultant library is often plagued with unwanted plasmids that have no inserts or multiple inserts. These species have to be eradicated to avoid tedious screening, especially when producing a mutant gene library. To overcome these problems, we modified the QuikChange(TM) protocol so that each plasmid carries a single insert. Although the QuikChange was originally developed for site-directed mutagenesis using complementary mutagenic oligonucleotide primers in whole plasmid PCR, we found that the protocol also worked for megaprimers consisting of hundreds of nucleotides. Based on this discovery, we used insert fragments, which we wanted to clone, as the primers in the QuikChange reaction. The resultant libraries were virtually free from species with no inserts or multiple inserts. The present method, which we designated MEGAWHOP (megaprimer PCR of whole plasmid), is thus ideal for creating random mutagenesis megalibraries.