High-throughput microtiter well-based bioluminometric genotyping of two single-nucleotide polymorphisms in the toll-like receptor-4 gene |
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Authors: | Iliadi Alexandra C Ioannou Penelope C Traeger-Synodinos Joanne Kanavakis Emmanuel Christopoulos Theodore K |
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Institution: | a Department of Chemistry, University of Athens, 15771, Athens, Greece b Medical Genetics, Athens University, St. Sophia’s Children’s Hospital, 11527, Athens, Greece c Department of Chemistry, University of Patras, 26500, Patras, Greece d Foundation for Research and Technology Hellas, Institute of Chemical Engineering and High Temperature Chemical Processes, P.O. Box 1414, 26504, Patras, Greece |
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Abstract: | 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. |
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Keywords: | Bioluminometric assay Genotyping Primer extension TLR4 |
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