Detection of Single-Base Substitutions in Amplified Fragments via Ligation of a Tandem of Short Oligonucleotides in Solution and on a Solid Carrier

Ligation of a tandem of short oligonucleotides was proposed for detecting single-base substitutions in amplified DNA fragments. An octamer–tetramer–octamer tandem was ligated on a 20-mer template with T4 DNA ligase. As shown with radiolabeled oligonucleotides, the efficiency and selectivity of ligation did not change with an octamer linked to a water-soluble carrier based on polyethylene glycol (MPEG), while ligation was somewhat lower with the octamer immobilized on methacrylate beads (DMEG). In both cases, polymer attachment improved the discrimination of 20-mer templates with single-base substitutions in the binding site for the tetramer or for the immobilized octamer. Tandems with a radiolabeled or biotinylated component were also efficiently ligated on amplified DNA fragments. The data obtained with DNA fragments of HIV-1 strains bru and rf demonstrate the possibility of reliable detection of single-base substitutions via ligation of a tandem and colorimetric detection of the immobilized ligation product with the streptavidin–alkaline phosphatase technique.