PCR-based overlap extension mutagenesis was applied to introduce a Thr(381) to Lys mutation in the alpha (2C)-adrenoceptor (alpha (2C) AR) coding sequence. This cDNA contains 71% G+C nucleotides and conventional PCR procedures were inefficient in generating a corresponding amplification product. Only 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 reagent and 5% (v/v) dimethylsulphoxide with the Advantage GC cDNA PCR kit yielded 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 profile for a series of alpha (2) AR ligands using [H-3]RX 821002.