Improvement of the sensitivity and resolution of PCR-SSCP analysis with optimized primer concentrations in PCR products.

Polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) is a widely utilized screening method for detection of mutations. In this method, the target sequence of interest is amplified by PCR and separated as single-stranded molecules by electrophoresis in a nondenaturing polyacrylamide gel (Orita et al. 1989). However, the sensitivity and resolution of PCR-SSCP can be influenced by many parameters, including size and GC content of the PCR product (Li et al. 2003); gel temperature during electrophoresis (Chen et al. 1995); buffer composition (e.g. ionic strength, pH; Kukita et al. 1997); buffer additives, mainly glycerol, formamide, polyethylene glycol (Markoff et al. 1997; Paccoud et al. 1998); gel matrix composition and concentration (Savov et al. 1992), as well as primer concentration in PCR product (Cai and Touitou 1993). It is generally considered that specific PCR amplification is important in detection of mutations by using PCR-SSCP (Orita et al. 1989; Li et al. 2003). Moreover, it has been noted that addition of either forward or reverse primer in PCR product may apparently alter SSCP banding patterns, which is presumably caused by the interaction between primer(s) and single-strand DNAs (ssDNA) (Almeida et al. 1998; Hennessy et al. 1998). Previously, Cai and Touitou (1993) demonstrated that the lowest primer concentration in PCR product affecting SSCP banding patterns is around 6 nM, and that primer-concentration-dependent intermediate SSCP patterns appear in unpurified PCR product amplified with regular amounts of primers (30–150 nM). In the present study, we used two identified PCR fragments of the promoter region in