Improved resolution of asymmetric-PCR SSCP products.

amide gel electrophoresis (PAGE) on a 180× 160× 1.5-mm 10% polyacrylamide gel. The separated DNA strands were subsequently visualized by staining with either EtdBr or silver (2). Separation of the DNA strands before electrophoresis using streptavidin paramagnetic beads consistently yielded single-stranded products upon SSCP analysis (Figure 1). Treatment of DNA strands with S1 nuclease before SSCP analysis confirmed the single-stranded nature of the stained bands (results not shown). When the SSCP technique using streptavidin-separated strands was applied to a number of rifampicin-resistant M. tuberculosis isolates, clear differences in mobility of at least one of the DNA strands, relative to the rifampicin-sensitive control, could be observed in all of the isolates (Figure 1). The rapid re-annealing of the DNA strands, which proved to be problematic in our studies, could be a consequence of the large quantities of DNA (0.5–1 μg) applied to the gel for a strong signal after EtdBr or silver staining. A higher concentration of DNA is likely to promote rapid re-annealing. Other detection methods, involving radioactive labeling of the PCR products for example, may not encounter such a problem because less DNA is needed to generate a strong signal after SSCP. The use of radioactivity has its disadvantages, but they are mostly in the hazardous nature of the use of radioisotopes. In this study, we have described an adaptation of the PCR-SSCP protocol that enables large amounts of DNA to be loaded onto the gel for subsequent EtdBror silver-stain detection and avoids the problem of single-strand reannealing. This approach also separates the two DNA strands into different lanes of the polyacrylamide gel, a process that is beneficial if the two strands have similar mobility that might confuse the analysis.