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), β-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.