Quadruple-allele chemiluminometric assay for simultaneous genotyping of two single-nucleotide polymorphisms.
We developed a rapid, simple, cost-effective and high sample-throughput method for the simultaneous detection of four alleles in single-nucleotide polymorphisms (SNPs). The method was applied to the simultaneous genotyping of two common SNPs within the TLR4 gene, the A896G and C1196T polymorphisms. The method consists of a single PCR of the region spanning the A896G and C1196T polymorphic sites, followed by a quadruple primer extension (PEXT) reaction in a single tube. A biotinylated nucleotide is incorporated in the extended primer. All four products are captured in streptavidin (SA)-coated microtiter wells and detected with a combination of four reporters, the photoprotein aequorin (AEQ) and the enzymes alkaline phosphatase (ALP), beta-galactosidase (GAL) and horseradish peroxidase (HRP). For each SNP, 46 individuals were genotyped. The accuracy of this method was confirmed by sequencing. The proposed quadruple-allele chemiluminometric assay provides an accurate, simple, rapid, reproducible and cost-effective method for high sample-throughput genotyping of single-nucleotide polymorphisms.
Elenis
Dimitrios S
DS
Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Greece 15771.
Ioannou
Penelope C
PC
Christopoulos
Theodore K
TK
D000483:Alleles; D005838:Genotype; D006801:Humans; D008163:Luminescent Measurements; D020641:Polymorphism, Single Nucleotide; D051197:Toll-Like Receptor 4 / Q000235:genetics*
C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4