Combined flash- and glow-type chemiluminescent reactions for high-throughput genotyping of biallelic polymorphisms

The difference in light-emission kinetics between the Ca(2+)-triggered bioluminescent reaction of the photoprotein aequorin (AEQ) and the alkaline phosphatase (ALP)-catalyzed chemiluminescent hydrolysis of dioxetane aryl phosphate substrates was exploited for the analysis of both alleles of biallelic polymorphisms in a single microtiter well. The genotyping of the IVS-1-110 locus of the human beta-globin gene was chosen as a model. Genomic DNA, isolated from whole blood, was first subjected to polymerase chain reaction using primers flanking the polymorphic site. A single oligonucleotide-ligation reaction employing two allele-specific probes, labeled with biotin and digoxigenin, and a common probe carrying a characteristic tail was then performed. The ligation products were captured in a microtiter well through hybridization of the tail with an immobilized complementary oligonucleotide. The products were detected by adding a mixture of streptavidin-aequorin complex and antidigoxigenin-alkaline phosphatase conjugate. AEQ was measured first by adding Ca(2+) and integrating the signal for 3s followed by the addition of the substrate for ALP. The ratio of the luminescence signals obtained from ALP and AEQ gives the genotype of each sample. The coefficient of variation of the dual assay ranged from 7 to 11% for each allele. The reproducibility of the ALP/AEQ signal ratio was about 14%. The proposed assay allows for many samples to be screened in parallel in a single microtiter plate, for single-nucleotide polymorphisms.