Europium-Labeled Oligonucleotidesto Detect Point Mutations: Applicationto P1Z

We describe a novel assay for detection of point mutations. The method combines the specificity and sensitivity of the potymerase chain reaction (PCR) and allele-specific oligonucleotides (ASO) with highly sensitive timeresolved 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-amplifled nucleic acid fragment. The hybrids formed were then collected onto streptavidincoated 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 underlyingthe design of the Eu-labeled ASO probes for the solution hybridization assay. The method was applied to the detection of the Z-mutation in the a1-antitrypsin gene. Evaluation of whole-blood samples spotted on Guthrie cards demonstrated successful accuracy of the method.