Quantitative and qualitative analysis of amplified DNA sequences by a competitive hybridization assay.
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.
Térouanne
B
B
INSERM Unité 58, Montpellier, France.
Balaguer
P
P
Boussioux
A M
AM
Nicolas
J C
JC
D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D008722:Methods; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction
D004247:DNA