Asymmetric PCR increases efficiency of melting peak analysis on the LightCycler

Objectives: To systematically analyze the effects of asymmetric PCR on LightCycler melting analyses of four different allelic-discrimination systems and to reduce an inconsistent non-specific melting peak observed during factor V Leiden genotyping. Design and methods: PCR amplifications and melting analyses were carried out with various oligonucleotide concentrations and ratios. To monitor the efficiency, calculated peak area values were compared after melting analyses. Results: Peak area values increased by a mean of 11.2-fold (range: 6 to 17) in case of an amplification primer ratio of 1:6.7 asymmetric PCR compared to symmetric primer conditions in four different SNP-genotyping systems. Using a complementary hybridization probe set for factor V Leiden genotyping, a converse amplification primer ratio was necessary for similar results. Conclusions: Asymmetric PCR resulting in the formation of higher amounts of target strands significantly increases the efficiency of LightCycler allelic-discrimination.