title: Rapid and reliable genotyping for the Toll-like receptor 4 A896G polymorphism using fluorescence-labeled hybridization probes in a real-time polymerase chain reaction assay.
abstract: BACKGROUND
  The Toll-like receptor 4 (TLR4) is involved in immune response to endotoxin as well as the pathogenesis of atherosclerosis. The TLR4 gene was shown to carry a single-nucleotide polymorphism (A896G). We developed a rapid-cycle polymerase chain reaction (PCR) which allows for rapid genotyping and, therefore, may be useful in clinical risk assessment.
  
  
  METHODS
  Fluorescently labeled oligonucleotide hybridization probes were designed for the LightCycler instrument. A PCR product was generated in a single reaction followed by melting point analysis. Ninety-three German Caucasians were genotyped. The interleukin-1beta (IL-1beta) response to endotoxin was assessed after whole blood stimulation with endotoxin according to TLR4 genotypes.
  
  
  RESULTS
  The method suggested by us is a time-saving technique requiring no additional manual steps. Frequencies of the A and G allele were 0.95 and 0.05, respectively. The study population was in Hardy-Weinberg equilibrium. The specificity of the method was confirmed by sequencing. The IL-1beta levels were lower in carriers of the G allele.
  
  
  CONCLUSIONS
  This PCR assay is a rapid and reliable technique for TLR4 genotyping.
authors.affiliation: Department of Anesthesia, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1100 DD Amsterdam, Netherlands. m.heesen@amc.uva.nl
authors.forename: Michael
authors.initials: M
authors.lastname: Heesen
authors.forename: Martina
authors.initials: M
authors.lastname: Wessiepe
authors.forename: Dagmar
authors.initials: D
authors.lastname: Kunz
authors.forename: Katarina
authors.initials: K
authors.lastname: Vasickova
authors.forename: Brunhilde
authors.initials: B
authors.lastname: Blomeke
mesh_terms: D005838:Genotype; D006801:Humans; D006824:Hybridization, Genetic; D007375:Interleukin-1 / Q000097:blood / Q000235:genetics; D008562:Membrane Glycoproteins / Q000032:analysis / Q000235:genetics*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D011956:Receptors, Cell Surface / Q000032:analysis / Q000235:genetics*; D015203:Reproducibility of Results; D013696:Temperature; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors
chemical_list: D007375:Interleukin-1; D008562:Membrane Glycoproteins; D015345:Oligonucleotide Probes; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors