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Rapid detection of the factor XIII Val34Leu (163 G-->T) polymorphism by real-time PCR using fluorescence resonance energy transfer detection and melting curve analysis.

Abstract
The Val34Leu polymorphism in the A subunit of blood coagulation factor XIII (FXIII-A) is located in the activation peptide, just three amino acids upstream of the thrombin cleavage site. The Val-->Leu replacement accelerates the rate of the proteolytic activation of FXIII and it seems to provide protection against myocardial infarction. Methods available for the assessment of the FXIII-A Val34Leu polymorphism are rather time-consuming, laborious and not easily applicable for large-scale studies. In this study a new method based on real-time PCR with fluorescence resonance energy transfer (FRET) detection and melting curve analysis was developed. The rapid, simple method was adapted to the widely used real-time PCR instrument, LightCycler (Roche Diagnostics). The results showed 100% coincidence with those obtained by the traditional PCR-restriction fragment length polymorphism (RFLP) assay and fluorescent DNA sequencing. Using this method, an allele frequency of 24.2% was obtained (n=113), which well agrees with the allele frequency obtained by PCR-RFLP on a different group of the same ethnic Hungarian population (25.9%).
AuthorsAmir H Shemirani, László Muszbek
JournalClinical chemistry and laboratory medicine (Clin Chem Lab Med) Vol. 42 Issue 8 Pg. 877-9 ( 2004) ISSN: 1434-6621 [Print] Germany
PMID15387436 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • DNA
  • Factor XIII
Topics
  • Alleles
  • DNA (chemistry, genetics)
  • Factor XIII (analysis, chemistry, genetics)
  • Fluorescence Resonance Energy Transfer (methods)
  • Gene Frequency
  • Humans
  • Hungary
  • Nucleic Acid Denaturation
  • Polymerase Chain Reaction (instrumentation, methods)
  • Polymorphism, Genetic
  • Polymorphism, Restriction Fragment Length
  • Temperature
  • Time Factors

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