Detection of single-base substitutions in amplified fragments via ligationof a tandem of short oligonucleotides in solution and on a solid carrier

Ligation of a tandem of short oligonucleotides was proposed for detecting s ingle-base substitutions in amplified DNA fragments. An octamer-tetramer-oc tamer tandem was ligated on a 20-mer template with T4 DNA ligase. As shown with radiolabeled oligonucleotides, the efficiency and selectivity of ligat ion did not change with an octamer linked to a water-soluble carrier based on polyethylene glycol (MPEG), while ligation was somewhat lower with the o ctamer immobilized on methacrylate beads (DMEG). In both cases, polymer att achment improved the discrimination of 20-mer templates with single-base su bstitutions in the binding site for the tetramer or for the immobilized oct amer. Tandems with a radiolabeled or biotinylated component were also effic iently ligated on amplified DNA fragments. The data obtained with DNA fragm ents of HIV-1 strains bnl and rf demonstrate the possibility of reliable de tection of single-base substitutions via ligation of a tandem and colorimet ric detection of the immobilized ligation product with the streptavidin-alk aline phosphatase technique.