Lauren Biotechnology, Nanjing 210019, China.
Anal Biochem. 2012 Oct 1;429(1):76-8. doi: 10.1016/j.ab.2012.06.027. Epub 2012 Jul 4.
Gene splicing and site-directed mutagenesis (SDM) are important to introduce desired sequences in target DNA. However, introducing mutations at multiple sites requires multiple steps of DNA manipulation, which is time-consuming and labor-intensive. Here, we present a rapid efficient gene splicing and multi-sited mutagenesis method that introduces mutations at two distant sites via sequential connection of DNA fragments by one-step overlap extension polymerase chain reaction (OE-PCR). This bottom-up approach for DNA engineering can be broadly used to study protein structure-function, to optimize codon use for protein expression, and to assemble genes of interest.
基因拼接和定点诱变（SDM）对于在目标 DNA 中引入所需序列非常重要。然而，在多个位点引入突变需要多次 DNA 操作，既耗时又费力。在这里，我们提出了一种快速高效的基因拼接和多位点诱变方法，通过一步重叠延伸聚合酶链反应（OE-PCR）顺序连接 DNA 片段，在两个遥远的位点引入突变。这种自下而上的 DNA 工程方法可广泛用于研究蛋白质结构-功能，优化用于蛋白质表达的密码子，并组装感兴趣的基因。
