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

摘要

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 deg C followed by quick chilling on ice and the addition of 1 M of a commercial GC Melt reagent and 5% (v/v) dimethylsuphoxide 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 [~3H] RX 821002.