Distinction of Weakly Homologous cDNA Amplificates by Single-Strand Conformation Polymorphism Analysis: Application to Guanylyl Cyclase Isozymes

Kurzfassung

By use of the polymerase chain reaction (PCR), uniform amplification products of 225 to 240 bp length were obtained from five cDNA clones representing different types of guanylyl cyclases. These short DNA double strands were differentiated by single-strand conformation polymorphism (SSCP), using polyacrylamide gel electrophoresis with the Pharmacia Phast-System. Following heat denaturation, the samples were separated on native polyacrylamide gels at different running temperatures. Nucleic acids on the gel were detected by an automated silver stain procedure. Using 7.5% homogeneous or 4-15% gradient polyacrylamide gels at a temperature of 12C, single-strand confor- mations of amplificates, representing three different particulate guanylyl cyclases and the two subunits of soluble guanylyl cyclase, were differentiated. The characteristic banding patterns resulting from dissimilar migration of the single-strand conformations were assigned to different guanylyl cyclase types. For the enzyme family of guanylyl cyclases, the feasibility of a combined PCR and electro- phoresis approach for analyzing the expression of related genes was demonstrated. This application of the PCR-SSCP technique provided a rapid and sensitive tool for the characterization of PCR products obtained with a common primer pair and suggested its use for investi-gating the tissue distribution of gene expression within a class of homologous proteins.