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.
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%).
Shemirani
Amir H
AH
Department of Clinical Biochemistry and Molecular Pathology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary.
Muszbek
László
L
D000483:Alleles; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D005176:Factor XIII / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005787:Gene Frequency; D006801:Humans; D006814:Hungary; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D013696:Temperature; D013997:Time Factors
D004247:DNA; D005176:Factor XIII