Excess PCR primers may dramatically affect SSCP efficiency.

SSCP analysis is one of the most commonly used techniques for screening of small genetic alterations and a very convenient alternative to direct sequencing. In this technique, PCR products are denatured and electrophoresed on non-denaturing polyacrylamide gels. Any change in the sequence theoretically causes a shift in the mobility of the analysed conformers. However, it is known (1) that certain changes remain undetected by SSCP analysis. We also failed (not shown) to discriminate between two 144 bp cathepsin D sequences which only differ by a C/T base substitution at position 224 (2), using the original (3) or modified (4) SSCP protocols on PCR products obtained from homozygous (T/T), heterozygous (C/T) and homozygous (C/Q breast cancer cell lines (MCF7, MDA-MB-231 and T47D respectively). We solved part of the problem (Figure la) by increasing the polyacrylamide concentration of the SSCP gels and by slowing down the speed of the electrophoretic run. The best results were obtained when gels were run at room temperature in 0.5 XTBE, 5% glycerol and 15% acrylamide for at least 6 h (the first 2 h at 50 volts and the end of run at 100 to 150 volts), and then silver stained (Kit Biorad, Paris, France). However, in these conditions where T/T genotypes were easily identified (lane 1), C/T heterozygotes (lane 2) and C/C homozygotes (lane 3) were still hardly or not at all separated. To identify migration of each of the two strands, we performed 'unbalanced' PCRs in which one of the two primer concentrations was lowered. Whereas lowering the downstream primer led to no amplification for unknown reasons, in SSCPs performed on unbalanced PCR products obtained using a downstream/upstream primer ratio of 12 (Figure la, lanes 4—6), all three genotype patterns were surprisingly different and bands migrated a little further than regular PCR products. SSCPs were discriminative when using unbalanced PCR. As the only difference between the two PCR protocols involved the primer concentrations, we hypothized that excess primers in samples at the end of amplifications interfered with the DNA strands, changing their conformation and subsequently their SSCP profiles. To address this question and test the effects of each PCR reactive on the SSCPs, we first performed a quick purification step of the 144 bp fragment: the specific DNA band was excised from the PCR control gel (5% polyacrylamide) and eluted in 50 yl H 2 O at 65 °C for 1 h. The eluted …