Quantitative and qualitative analysis of amplified DNA sequences by a competitive hybridization assay

Abstract

The presence of allelic sequence variations in DNA fragments can be easily detected by measuring the extent of DNA strand exchange between test double-stranded PCR products (target) and labeled standard double-stranded PCR products (probe). Under selected hybridization conditions, sequences identical to the probe decreased the formation of double-labeled hybrid, whereas differing sequences were not efficient enough to compete with the regeneration of the probe. A single base substitution in the target DNA increased the percentage of remaining double-labeled probe. A general procedure involving denaturation and hybridization in solution under different temperature conditions or using different probes enabled sequence identification. The degree of regeneration of double-labeled probe was determined using a bioluminescent assay. We evaluated the specificity of this method with two probes (108 and 131 bp) and several targets with different base substitutions.