Non-isotopic detection of single-stranded conformation polymorphisms using ethidium bromide/UV light.

Abstract

In the course of searching for single base substitutions resulting in polymorphisms or point mutations within the adenomatous Polyposis coli (APC) gene we established an alternative non-radioactive procedure to detect single-stranded conformation polymorphisms (SSCP) of polymerase chain reaction (PCR) products. A fast and inexpensive SSCP assay system is described in detail with general application allowing the non-isotopic identification of single base alterations of PCR products. This was achieved by combining heat denaturation of PCR products resulting in a high yield of single-stranded DNA and ethidium bromide staining of separated DNA strands in non-denaturing polyacrylamide vertical standard gels. The validity of the non-radioactive procedure described here is demonstrated by the identification of allele-specific SSCP patterns derived from intragenic polymorphisms of the APC gene.