Improvement of SSCP analysis by use of denaturants.

Single-strand conformation polymorphism (SSCP) analysis is a simple and quick method for mutation detection. It depends on the separation of single-stranded (ss)DNA fragments in non-denaturing polyacrylamide gels. On occasion, the ssDNA bands can be very diffuse or appear against a heavily smeared background. Here, we demonstrate that the addition of denaturants, urea or formamide, to the gels can sharpen the bands of ssDNA fragments and clear the background that might otherwise obscure bands that are diagnostic of a polymorphism. We have recently studied seven new polymorphisms in polymerase chain reaction (PCR)-amplified fragments from the human PGM1 locus (7). Two fragments, designated M3 and N1, initially gave broad or diffuse bands and heavy background smearing in the SSCP gels (Figures 1A and 2A), despite the demonstration of single intensely stained DNA bands on electrophoresis in agarose gel (data not shown), and native and denaturing polyacrylamide gels (Figure 3). We hypothesized that the smearing was due to the occurrence of multiple conformations of ssDNA with similar, but not identical, electrophoretic mobilities. We theorized that a mildly denaturing environment in the gel would make the multiple conformations move with a more uniform mobility by reducing their number, and this could clear the background smearing and improve the resolution. With the M3 fragment (54% GC content), a pronounced improvement in the resolution of the ssDNA bands was obtained with 10% (wt/vol) urea or 10% (vol/vol) formamide in the gel though the relative mobilities of the variant bands were reversed (Figure 1B). Slight improvement in resolution was also obtained with 10% (vol/vol) dimethyl sulfoxide (DMSO), but here the bands retained their original relative mobilities and were still somewhat blurred. With the N1 fragment (77% GC content), considerable improvement in the resolution of the bands was obtained with urea or formamide, but not DMSO. The gel concentration of formamide was titrated in 5% incremental steps from 10%–30% to find the optimum conditions (Figure 2). In the absence of formamide, the bands of both the C and D alleles were very diffuse, and the C band was in the center of the smeared regions associated with the A and B bands. Therefore, it was not possible to type the C and D alleles. As the formamide concentration was increased, three effects were evident: (i) the sharpening of the C and D bands, (ii) the clearing of the background smearing and (iii) the appearance of some additional bands. At a formamide concentration above 20%, the relative mobilities of the A and B bands were reversed. The use of denaturants in SSCP gels was first reported by Glavac and Dean (2) when they were exploring optimization strategies in SSCP analysis. Three other groups also reported the use of formamide (but not urea) in SSCP analysis (3,5,6), and one of these claimed that genotyping was easier in the presence of formamide (3). Recently, addition of sodium dodecyl sulfate (SDS) (1) or polyethylene glycol (PEG) (4) in the gel has also been found to enhance the technique. The use of strong denaturants in the gels seems to contradict the basic working principles of the SSCP technique. However, we have shown that the incorporation of denaturants in SSCP gels can lead to significant enhancement of resolution, clearance of background smearing and changes in relaBenchmarks