An improved PCR-mutagenesis strategy for two-site mutagenesis or sequence swapping between related genes.
The QuikChangeTM protocol is one of the simplest and fastest methods for site-directed mutagenesis, but introduces mutations at only one site at a time, and requires two HPLC-purified complementary oligonucleotides. Here, we describe that this method can be used with non-overlapping oligonucleotides. By doing this, two separate sites can be mutagenised simultaneously, or money can be saved by using a second 'standard' oligonucleotide. By a further modification, we have also used the QuikChangeTM approach to exchange DNA sequences between closely related genes.
Kirsch
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RD
The Babraham Institute, Babraham, Cambridge CB2 4AT, UK.
Joly
E
E
D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000302:isolation & purification; D010957:Plasmids; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D014162:Transfection
D017931:DNA Primers; D009838:Oligodeoxyribonucleotides; D004247:DNA