Clinical Chemistry, Vol 41, 413-418, Copyright © 1995 by American Association for Clinical Chemistry

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

TS Hansen, NE Petersen, A Iitia, O Blaabjerg, P Hyltoft-Petersen and M Horder
Department of Clinical Chemistry, Odense University Hospital, Denmark.

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 simultaneous 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 simple to perform, is very reliable, and can be partly automated, we conclude that it is well-suited for analysis in a routine laboratory.

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