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 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.
Hansen
T S
TS
Department of Clinical Chemistry, Odense University Hospital, Denmark.
Petersen
N E
NE
Iitiä
A
A
Blaabjerg
O
O
Hyltoft-Petersen
P
P
Hørder
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D019389:Cytochrome P-450 CYP2D6; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D011088:DNA Ligases / Q000378:metabolism; D004252:DNA Mutational Analysis; D005063:Europium; D005838:Genotype; D006801:Humans; D006899:Mixed Function Oxygenases / Q000235:genetics*; D009841:Oligonucleotides / Q000378:metabolism*; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012493:Samarium; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature
D009841:Oligonucleotides; D004364:Pharmaceutical Preparations; D012493:Samarium; D005063:Europium; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019389:Cytochrome P-450 CYP2D6; D011088:DNA Ligases