Nucleic Acids Research, 1990, Vol. 18, No. 24 7433-7438
© 1990
Articles |
Site-specific mutagenesis using asymmetric polymerase chain reaction and a single mutant primer
Laboratory of the Howard Hughes Medical Institute, Division of Hematology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School Boston, MA 02115, USA
*To whom correspondence should be addressed
Received March 23, 1990. Accepted April 24, 1990.
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, ß-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. Singie 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 otfers a rapid means for producing competitive templates for use in quantitative PCR.
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