A dual label oligonucleotide ligation assay for detection of the CYP2C19*1, CYP2C19*2, and CYP2C19*3 alleles involving time-resolved fluorometry.

CYP2C19 (S-mephenytoin hydroxylase) is a polymorphically expressed enzyme. Currently, two defective alleles are known--CYP2C192 and CYP2C193. The authors have developed an oligonucleotide ligation assay to detect these two alleles. This assay combines the hybridization of one common, biotinylated capture probe and two allele-specific probes to the target DNA, with the ability of a DNA ligase to distinguish mismatched nucleotides. The probes are only ligated if they are base paired correctly to the target strand. The biotin is bound to streptavidin, and all DNA not covalently bound to the biotin-labeled capture probe, is removed in a washing procedure. The allele-specific probes are labeled with either europium or samarium, and their emission can be measured simultaneously. The ratio between the emission separates the genotypes. This method was applied on DNA from 19 whites and 21 Vietnamese living in Denmark. All genotypes determined by the assay were consistent with the results from restriction enzyme cleavage. There were 12 poor metabolizers; 10 homozygous CYP2C192/CYP2C192, one heterozygous CYP2C192/CYP2C193, and one heterozygous CYP2C191/CYP2C192. The authors conclude that this assay is well-suited for a high throughput of samples in a routine laboratory. The finding of an apparently heterozygous CYP2C191/CYP2C192 poor metabolizer, confirms that there are still unknown mutations in CYP2C19.