Site-specific mutagenesis using asymmetric polymerase chain reaction and a single mutant primer.
AUTOR(ES)
Perrin, S
RESUMO
A method is described for preparing site-specific mutants using a polymerase chain reaction (PCR) based protocol. The protocol requires a single mutant primer, and has been used to introduce mutations into DNA fragments ranging in size from 200 bp to 1569 bp in length in the GM-CSF, beta-actin, human growth hormone and erythropoietin genes. Sequence analysis of PCR derived mutant fragments shows an error rate of less than one bp change per 1500 bp incorporated. Single base pair mutations have been introduced into these genes which create unique restriction sites. We demonstrate that these mutant templates may be used for competitive PCR to quantitate mRNA and DNA. This method thus offers a rapid means for producing competitive templates for use in quantitative PCR.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=332882Documentos Relacionados
- Conditional mutagenesis using site-specific recombination in Plasmodium berghei
- A system for site-specific mutagenesis of the photosynthetic reaction center in Rhodopseudomonas viridis.
- Site-directed mutagenesis using a double-stranded DNA fragment as a PCR primer.
- Rapid and efficient site-specific mutagenesis without phenotypic selection.
- Rapid genome walking: a simplified oligo-cassette mediated polymerase chain reaction using a single genome-specific primer