An improved PCR-based method for site directed mutagenesis using megaprimers.
An improved protocol for site-directed mutagenesis based on the two-step polymerase chain reaction (PCR) megaprimer method is described. Compared to previously published protocols, the protocol described in this article ensures consistently a success rate of at least 85% with essentially no introduction of unwanted secondary mutations. The essential features of this protocol include an optimization of the template-primer amounts and ratio that allows the use of a reduced number of PCR cycles and the use of proof-reading thermostable DNA polymerases.
Brøns-Poulsen
J
J
Department of Molecular Biology, Odense University, Odense, Denmark.
Petersen
N E
NE
Hørder
M
M
Kristiansen
K
K
D017931:DNA Primers / Q000737:chemistry / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase; D006801:Humans; D011163:Hydroxymethylbilane Synthase / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D011163:Hydroxymethylbilane Synthase; C072146:Pfu DNA polymerase; D004259:DNA-Directed DNA Polymerase