Detection of a point mutation using short oligonucleotide probes in allele-specific hybridization

Two nonradioactive and simple procedures were developed to detect the A985G point mutation that causes medium-chain acyl-CoA deficiency. In both of these assays, short oligonucleotide probes were used in allele-specific hybridization combined with DNA amplification. The lower limit for a useful probe was found to be between 9 and 12 base pairs. Time-resolved fluorometry was utilized as the label technology and microtitration plates as the solid support. In one of the assay formats, probes labeled with europium and samarium chelates were used to simultaneously detect the mutant and normal alleles from the same hybridization reaction. In addition, the discrimination efficiency of different probes was characterized by cross-reactivity determinations and by measuring affinities of the probes towards fully complementary as well as towards mismatch-forming target oligonucleotides. All of the 80 coded patient samples analyzed were correctly typed in both of the assay formats used.