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

Amir H. Shemirani, L. Muszbek

Research output: Contribution to journalArticle

20 Citations (Scopus)

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%).

Original languageEnglish
Pages (from-to)877-879
Number of pages3
JournalClinical Chemistry and Laboratory Medicine
Volume42
Issue number8
DOIs
Publication statusPublished - 2004

Fingerprint

Factor XIII
Fluorescence Resonance Energy Transfer
Polymorphism
Freezing
Real-Time Polymerase Chain Reaction
Melting
Gene Frequency
Restriction Fragment Length Polymorphisms
Chemical activation
Polymerase Chain Reaction
DNA Sequence Analysis
Ethnic Groups
Thrombin
Assays
Myocardial Infarction
Amino Acids
Peptides
DNA
Population

Keywords

  • Factor XIII
  • Fluorescence resonance energy transfer
  • Gene polymorphism
  • Real-time PCR

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

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title = "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{\%}).",
keywords = "Factor XIII, Fluorescence resonance energy transfer, Gene polymorphism, Real-time PCR",
author = "Shemirani, {Amir H.} and L. Muszbek",
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AU - Shemirani, Amir H.

AU - Muszbek, L.

PY - 2004

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N2 - 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%).

AB - 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%).

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