重叠延伸PCR对DNA片段进行定点双突变

探讨如何利用重叠延伸PCR对同一靶DNA片段中的两个不同位点实施联合突变。先用野生型DNA作模板,通过一轮重叠延伸PCR,获得突变一个预期位点的DNA片段,再用此突变DNA片段作模板,通过另一轮重叠延伸PCR获得两个预期位点均突变的DNA片段。重叠延伸PCR能对DNA片段进行双突变甚至多点突变,具有简便、快速、经济等特点,在阐明基因的调控机理、改造蛋白质结构等分子生物学领域中具有极大的应用价值。

Simultaneous Introduction of Two Separate Site-Specific Mutations in a DNA Fragment Using Overlap Extension PCR

How to create two separate site-specific mutagenesis in a DNA fragment using overlap extension PCR was explored.First,the DNA fragment containing the one of two predetermined mutagenic sites was obtained by overlap extension PCR using the wild DNA as template,then the DNA fragment containing two predetermined mutagenic sites was obtained by another round of overlap extension PCR using the single-site mutagenic DNA.The overlap extension PCR is a simple,rapid and economic method to introduce two separate point mutations or multiple separate point mutations in the DNA fragment.It can be applied to clarify the mechanism of gene regulation and modify the structure of the proteins,thus has the potential application value in the field of molecular biology.