DescriptionSSCPs are DNA fragments of about 200-800 bp amplified by PCR using specific primers of 20-25 bp. Gel electrophoresis of single-strand DNA is used to detect nucleotide sequence variation among the amplified fragments. The method is based on the fact that the electrophoretic mobility of single-strand DNA depends on the secondary structure (conformation) of the molecule, which is changed significantly with mutation. Thus, SSCP provides a method to detect nucleotide variation among DNA samples without having to perform sequence reactions. In SSCP the amplified DNA is first denatured, and then subject to non-denaturing gel electrophoresis. Related techniques to SSCP are Denaturing Gradient Gel Electrophoresis (DGGE) that uses double stranded DNA which is converted to single stranded DNA in an increasingly denaturing physical environment during gel electrophoresis, and Thermal Gradient Gel Electrophoresis (TGGE) which uses temperature gradients to denature double stranded DNA during electrophoresis.
StrengthsAdvantages of SSCP are the codominance of alleles and the low quantities of template DNA required due to the fact that the technique is PCR-based.
WeaknessesDrawbacks include the need for sequence data to design PCR primers and the necessity of highly standardized electrophoretic conditions in order to obtain reproducible results. Furthermore, some mutations may remain undetected, and hence absence of mutation cannot be proven.
ApplicationsSSCPs have been used to detect mutations in genes using gene sequence information for primer construction.
PCR-SSCP: a method for detection of mutations Hayashi, K. (1992). Genetic Analysis: Techniques and Applications, 9: 73-79. doi:10.1016/1050-3862(92)90001-L.