Single-stranded Conformation Polymorphism (SSCP)

Originally described by Orita et al. in 1989,58 SSCP has been widely applied in the field of human genetics, where it has been used both for SNP detection and for SNP discovery,59 although it is generally preferred for the former application. The essence is to denature PCR products to make them single stranded and then to separate them by gel electrophoresis under non-denaturing conditions. In the absence of strong denaturants, a single-stranded fragment of DNA will adopt a unique and specific three-dimensional conformation as it attempts to fold into the most stable structure. The mutated form will adopt a different conformation to its wild-type counterpart. The differences in the conformers can be assessed on electrophoresis gels, where a heterozygous sample normally displays four bands, one for each denatured strand, and homozygotes normally display two bands (Figure 1). For mutation detection, the resolving power of the technique is improved by scanning PCR fragments that are less than 300 base pairs in length. Provided that PCR-SSCP analysis is conducted under appropriate conditions, for example by repeating each electrophoresis experiment at two running temperatures or by varying the amount of mild denaturant included in the gel (such as formamide), it has been shown to be an efficient approach to discovering new mutations. Initially SSCPs were detected using autoradiography of radiolabelled PCR products, followed later by silver staining to visualise unlabelled DNA fragments. A semi-automated method, PLACE-SSCP, has been developed in which the products of the PCR are labelled with fluorescent dyes and analysed by capillary electrophoresis under SSCP conditions. As with many other mutation detection techniques, SSCP analysis is amenable tomul tiplexed PCR formats where several PCR products labelled with different fluorophores are analysed in the same electrophoretic lane.

Figure 1 Principle of SSCP analysis. In the presence of heat and formamide, the dsDNA PCR products from a heterozygote are denatured for several minutes to form single-stranded DNA (ssDNA). Immediately before loading on to a non-denaturing electrophoresis gel, the samples are cooled on ice to encourage the formation of ssDNA conformers. Each conformer has a unique electrophoretic mobility as shown on the gel diagram.