Development and application of non-radio isotropic PCR-SSCP analysis for genetic diagnosis

We applied a new and powerful technique for genetic diagnosis, so called reverse transcript-polymerase chain reaction-single strand conformation polymorphism (RT-PCR-SSCP) without using radio labeled materials (non-RI). The principle of this method is mobility shift analysis of single-stranded DNAs on neutral polyacrylamide gel electrophoresis to detect DNA polymorphisms. Like restriction fragment length polymorphisms (RFLPs), SSCPs were found to be allelic variants of true Mendelian traits. In this study, this method was used to determine the mutations of the LDL receptor or insulin receptor. We found new mutations in Exon 14 of the LDL receptor, and a patient had a new missense mutation in which Asn461 was substituted for Thr461 in the alpha-subunit of the insulin receptor. Our findings indicate that this analysis can be successfully used to not only rapidly and easily but also safely screen mutation containing exons in large genes, compared to the conventional Southern blotting analysis. Moreover, this analysis has the advantage over the RFLP analysis that it can detect DNA polymorphisms and point mutations at a variety of positions in the DNA fragments. This "non-RI" RT-PCR-SSCP analysis is expected to be useful as a routine examination for genetic diagnosis in the ordinary laboratory.