A HIGHLY EFFICIENT PROCEDURE FOR SITE-SPECIFIC MUTAGENESIS OF FULL-LENGTH PLASMIDS USING VENT DNA-POLYMERASE

被引：82

作者：

BYRAPPA, S ^{[1
]}

GAVIN, DK ^{[1
]}

GUPTA, KC ^{[1
]}

机构：

[1] RUSH PRESBYTERIAN ST LUKES MED CTR,DEPT IMMUNOL MICROBIOL,CHICAGO,IL 60612

来源：

GENOME RESEARCH | 1995年 / 5卷 / 04期

关键词：

D O I：

10.1101/gr.5.4.404

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Careful titration of Vent polymerase activity allows efficient amplification of full-length plasmids (12 kb). The high processivity and fidelity of this enzyme made oligonucleotide-directed site-specific mutagenesis of plasmids a straight-forward process. Using only two primers, a mutagenic and a complementary, single-base mutants of recombinant plasmids were obtained consistently with > 90% efficiency from a single round of PCR. This procedure also made site-specific deletion, insertion, and several bases mutagenesis facile and efficient.

引用

页码：404 / 407

页数：4

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