Single-strand conformational polymorphism analysis: basic principles and routine practice.

Abstract

Single-strand conformational polymorphism (SSCP) analysis is a simple and sensitive technique for mutation detection and genotyping. The principle of SSCP analysis is based on the fact that single-stranded DNA has a defined conformation. Altered conformation due to a single base change in the sequence can cause single-stranded DNA to migrate differently under nondenaturing electrophoresis conditions. Therefore wild-type and mutant DNA samples display different band patterns. SSCP analysis involves the following four steps: (1) polymerase chain reaction (PCR) amplification of DNA sequence of interest; (2) denaturation of double-stranded PCR products; (3) cooling of the denatured DNA (single-stranded) to maximize self-annealing; (4) detection of mobility difference of the single-stranded DNAs by electrophoresis under non-denaturing conditions. Several methods have been developed to visualize the SSCP mobility shifts. These include the incorporation of radioisotope labeling, silver staining, fluorescent dye-labeled PCR primers, and more recently, capillary-based electrophoresis. Silver staining is simple, rapid, and cost-effective, and can be routinely performed in clinical laboratories. The use of SSCP analysis to discover and genotype single-nucleotide polymorphisms (SNPs) has been widely applied to the genetics of hypertension, including both monogenic (e.g., Liddle's syndrome) and polygenic disorders (e.g., essential hypertension).