A new approach to 'megaprimer' polymerase chain reaction mutagenesis without an intermediate gel purification step.
BACKGROUND
Site-directed mutagenesis is an efficient method to alter the structure and function of genes. Here we report a rapid and efficient megaprimer-based polymerase chain reaction (PCR) mutagenesis strategy that by-passes any intermediate purification of DNA between two rounds of PCR.


RESULTS
The strategy relies on the use of a limiting concentration of one of the flanking primers (reverse or forward) along with the normal concentration of mutagenic primer, plus a prolonged final extension cycle in the first PCR amplification step. This first round of PCR generates a megaprimer that is used subsequently in the second round of PCR, along with the second flanking primer, but without the intermediate purification of the megaprimer. The strategy has been used successfully with four different plasmids to generate various mutants.


CONCLUSIONS
This strategy provides a very rapid, inexpensive and efficient approach to perform site-directed mutagenesis. The strategy provides an alternative to conventional megaprimer based site-directed mutagenesis, which is based on an intermediate gel purification step. The strategy gives a high frequency of mutagenesis.
Tyagi
Rajiv
R
Department of Biochemistry and Molecular Biology, The University of Queensland, Brisbane, Qld 4072, Australia. r.tyagi@mailbox.uq.edu.au
Lai
Richard
R
Duggleby
Ronald G
RG
D000429:Alcohol Oxidoreductases / Q000235:genetics; D004247:DNA / Q000302:isolation & purification; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D050540:Ketol-Acid Reductoisomerase; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D004247:DNA; D000429:Alcohol Oxidoreductases; D050540:Ketol-Acid Reductoisomerase