Asymmetric PCR increases efficiency of melting peak analysis on the LightCycler.
OBJECTIVE
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.


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.
Szilvási
Anikó
A
Laboratory of Molecular Genetics, Institute of Hematology and Immunology, National Medical Center, Diószegi út 64, Budapest H-1113, Hungary.
Andrikovics
Hajnalka
H
Kalmár
Lajos
L
Bors
András
A
Tordai
Attila
A
D016375:Antisense Elements (Genetics); D004252:DNA Mutational Analysis / Q000379:methods*; D015342:DNA Probes; D031541:Fluorescence Resonance Energy Transfer; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics
D016375:Antisense Elements (Genetics); D015342:DNA Probes