Asymmetric PCR increases efficiency of melting peak analysis on the LightCycler

Anikó Szilvási, Hajnalka Andrikovics, Lajos Kalmár, András Bors, Attila Tordai

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)727-730
Number of pages4
JournalClinical Biochemistry
Volume38
Issue number8
DOIs
Publication statusPublished - Aug 1 2005

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Keywords

  • Allelic-discrimination
  • Asymmetric PCR
  • Hybridization probes
  • LightCycler

ASJC Scopus subject areas

  • Clinical Biochemistry

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