INCREASED RELIABILITY OF SELECTIVE PCR BY USING ADDITIONALLY MUTATED PRIMERS AND A COMMERCIAL TAQ DNA-POLYMERASE ENHANCER

被引：7

作者：

DEMILITO, A ^{[1
]}

CATUCCI, M ^{[1
]}

IANNELLI, F ^{[1
]}

ROMANO, L ^{[1
]}

ZAZZI, M ^{[1
]}

VALENSIN, PE ^{[1
]}

机构：

[1] UNIV SIENA,DIPARTIMENTO BIOL MOLEC,SEZ MICROBIOL,I-53100 SIENA,ITALY

来源：

MOLECULAR BIOTECHNOLOGY | 1995年 / 3卷 / 02期

关键词：

SELECTIVE POLYMERASE CHAIN REACTION; HUMAN IMMUNODEFICIENCY VIRUS; REVERSE TRANSCRIPTASE; MUTATION;

D O I：

10.1007/BF02789112

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：

A reliable selective PCR procedure that combines the use of additionally mutated primers with the specificity-enhancing properties of a commercial preparation (Perfect Match, Stratagene) is described. The human immunodeficiency virus type 1 pol gene point mutations known to confer in vitro resistance to azidothymidine were examined as a model for optimization of the assay. The usual strategy of deliberately introducing an additional mismatch 1 residue from the 3' end in the wild-type and mutant primers did not allow reproducible discrimination between wild-type and mutant target sequences. Addition of minimal amounts of Perfect Match to the same PCR mixtures resulted in a significantly enlarged range of selective annealing temperatures, providing a valuable and cost-effective means for reliable detection of known mutations by selective PCR.

引用

页码：166 / 169

页数：4

共 8 条

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