High-throughput microtiter well-based bioluminometric genotyping of two single-nucleotide polymorphisms in the toll-like receptor-4 gene.
Toll-like receptors (TLRs) play a fundamental role in pathogen recognition and activation of innate immunity. Genetic variations in TLR have been associated with reduced host immune response to TLR ligands. We developed a rapid, simple and cost-effective method for identification of two common single-nucleotide polymorphisms (SNPs) within TLR4 gene in a high-throughput format. The method consists of a single polymerase chain reaction of the region spanning the A896G and C1196T polymorphic sites, followed by two primer extension reactions at each site using primers that carry a (dA)(24) segment at the 5' end. A biotinylated nucleotide is incorporated in the extended primer. The products are captured in microtiter wells coated with streptavidin and detected using a (dT)(30)-conjugated photoprotein aequorin. A total of 209 individuals were genotyped for each SNP. The A896G and C1196T polymorphisms were found to be in linkage disequilibrium; 186 individuals (89%) were wild-type homozygous (A/A or C/C), 22 (10.5%) were heterozygotes (A/G or C/T), and 1 (0.5%) was homozygous for the mutation (G/G or T/T). The accuracy of this method was confirmed by sequencing. The newly developed method may be useful for association studies of these two SNPs with several diseases.
Iliadi
Alexandra C
AC
Department of Chemistry, University of Athens, 15771, Athens, Greece.
Ioannou
Penelope C
PC
Traeger-Synodinos
Joanne
J
Kanavakis
Emmanuel
E
Christopoulos
Theodore K
TK
D019921:Biotinylation; D005787:Gene Frequency; D005838:Genotype; D006801:Humans; D015810:Linkage Disequilibrium; D049449:Luminescence; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results; D051197:Toll-Like Receptor 4 / Q000235:genetics*
D051197:Toll-Like Receptor 4