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

AUTOR(ES)

Rust, S

RESUMO

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.

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