Asymmetric PCR increases efficiency of melting peak analysis on the LightCycler

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.

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.

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.

Asymmetric PCR resulting in the formation of higher amounts of target strands significantly increases the efficiency of LightCycler allelic-discrimination.