Gene splicing and mutagenesis by PCR-driven overlap extension
Gene splicing and mutagenesis by PCR-driven overlap extension
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DOI：
10.1038/nprot.2007.132
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Pease, Larry R.
中科院分区：
文献类型：
--
作者：
Heckman, Karin L.;Pease, Larry R.
通过PCR延伸重叠基因片段是用于定点突变和基因剪接的简单、通用的技术。初始PCR产生重叠的基因片段，然后将其用作另一个PCR的模板DNA以产生全长产物。内部引物在中间区段上产生重叠的互补3'末端，并引入核苷酸取代、插入或缺失以用于定点诱变或用于基因剪接，编码在相邻基因区段的接合处发现的核苷酸。这些中间产物的重叠链在随后的PCR中在该3'区域杂交，并延伸以产生通过侧翼引物扩增的全长产物，所述侧翼引物可包括用于将产物插入表达载体中以用于克隆目的的限制性酶位点。通过这种方法，突变体或嵌合基因的高效产生可以容易地用标准实验室试剂在大约1周内完成。
Extension of overlapping gene segments by PCR is a simple, versatile technique for site-directed mutagenesis and gene splicing. Initial PCRs generate overlapping gene segments that are then used as template DNA for another PCR to create a full-length product. Internal primers generate overlapping, complementary 3' ends on the intermediate segments and introduce nucleotide substitutions, insertions or deletions for site-directed mutagenesis, or for gene splicing, encode the nucleotides found at the junction of adjoining gene segments. Overlapping strands of these intermediate products hybridize at this 3' region in a subsequent PCR and are extended to generate the full-length product amplified by flanking primers that can include restriction enzyme sites for inserting the product into an expression vector for cloning purposes. The highly efficient generation of mutant or chimeric genes by this method can easily be accomplished with standard laboratory reagents in approximately 1 week.