Optimisation of overlap extension PCR-based mutagenesis of a GC-rich DNA template: application to the human alpha(2C)-adrenoceptor cDNA

PCR-based overlap extension mutagenesis was applied to introduce a Thr(381) to Lys mutation in the alpha (2C)-adrenoceptor (alpha (2C) AR) coding sequ ence. This cDNA contains 71% G+C nucleotides and conventional PCR procedure s were inefficient in generating a corresponding amplification product. Onl y the combined use of a pre-PCR denaturation step at 100 degreesC followed by quick chilling on ice and the addition of 1 M of a commercial GC Melt re agent and 5% (v/v) dimethylsulphoxide with the Advantage GC cDNA PCR kit yi elded efficient amplification during the three successive PCR steps of the overlap extension procedure. Transient expression of the mutant Thr(381)Lys alpha (2C) AR in Cos-7 cells was performed to determine the binding profil e for a series of alpha (2) AR ligands using [H-3]RX 821002.