Mutagenically separated PCR (MS-PCR): a highly specific one step procedure for easy mutation detection

Abstract

With increasing knowledge about the causal role of genetic defects in clinical diseases the necessity is apparent to have procedures for rapid diagnosis of point mutations. We developed a PCR-based technique, whereby both normal and mutant alleles can be amplified in the same reaction tube, using different length allele-specific primers. Furthermore the allele-specific primers introduce additional deliberate differences into the allelic PCR-products that drastically reduce crossreactions in subsequent cycles. This mutagenesis separates the amplification reactions of the alleles performed in the same tube. Subsequent identification of the PCR-products is done by gel electrophoresis and shows at least one of the two allelic products. Therefore, in addition to simple handling, MS-PCR provides a within-assay quality control for the exclusion of false negative results. The feasibility of this technique has been tested using six different mutations. The high sensitivity of MS-PCR also allows screening for mutation carriers in pooled DNA samples.