EUROPIUM-LABELED OLIGONUCLEOTIDES TO DETECT POINT MUTATIONS - APPLICATION TO PL-Z ALPHA-1-ANTITRYPSIN DEFICIENCY

We describe a novel assay for detection of point mutations. The method combines the specificity and sensitivity of the polymerase chain reac tion (PCR) and allele-specific oligonucleotides (ASO) with highly sens itive time-resolved fluorometry. ASO probes differing by a single base substitution and labeled with europium (Eu) chelates were hybridized in solution simultaneously with a biotinylated oligomer to a PCR-ampli fied nucleic acid fragment. The hybrids formed were then collected ont o streptavidin-coated microtitration wells. Subsequently, the hybrids were washed under stringent conditions and the remaining ASO probe was measured in a time-resolved fluorometer. We discuss the strategy unde rlying the design of the Eu-labeled ASO probes for the solution hybrid ization assay. The method was applied to the detection of the Z-mutati on in the alpha1-antitrypsin gene. Evaluation of whole-blood samples s potted on Guthrie cards demonstrated successful accuracy of the method .