Robust nonradioactive oligonucleotide ligation assay to detect a common point mutation in the CYP2D6 gene causing abnormal drug metabolism

A new nonradioactive oligonucleotide ligation assay for the detection of a common point mutation in the CYP2D6 gene is presented. The assay takes advantage of simul-taneous time-resolved fluorescence measurements of lanthanide-labeled probes and the specificity of T4-DNA ligase in combination with the polymerase chain reaction. This strategy makes it possible to perform the assay using both the wild-type-specific and mutant-specific probes simultaneously, securing an internal control in each reaction. We show that the allele-specific ligation part of the assay can be performed with great accuracy over a wide range of temperatures, salt concentrations, and T4-DNA ligase concentrations. This eliminates the risk of false-positive or false-negative reactions due to variations in these factors. Because the assay is simpl