GENERATION OF A PLASMID VECTOR FOR DELETION CLONING BY RAPID MULTIPLE SITE-DIRECTED MUTAGENESIS

被引:3
作者
BHAT, KS
机构
[1] National Institutes of Health, Rocky Mountain Laboratories, Hamilton, MT 59840
关键词
DNA SEQUENCING; ESCHERICHIA-COLI; PCR; RESTRICTION ENDONUCLEASE; SUBCLONING;
D O I
10.1016/0378-1119(93)90177-5
中图分类号
Q3 [遗传学];
学科分类号
071007 ; 090102 ;
摘要
The construction of a new plasmid vector, devoid of all Mbol (GATC) and TspEI(AATT) restriction sites, is described. The lack of these two frequent-cutting restriction sites is a unique feature among plasmids. This new plasmid, pBRkanf1-, allows selective fragmentation of a cloned insert. As a result, the vector offers an alternative strategy to create overlapping and sequentially deleted subclones. In addition, the construction of the new plasmid required the development of a rapid and accurate multiple site-directed mutagenesis procedure. The mutagenesis method uses a combination of DNA amplification and chain extension by DNA polymerase. By this method, mutations are created progressively from one end of a DNA molecule to the other.
引用
收藏
页码:83 / 87
页数:5
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