HYBRIDIZATION-AT-TAILING (HYBRAT) METHOD FOR SENSITIVE AND STRAND-SPECIFIC DETECTION OF DNA AND RNA

Delta Tth DNA polymerase catalyzed polymerization of dATP and dTTP int o a high-molecular-weight d(A-T) copolymer using oligo-d(A-T) as the t emplate/primer (Hanaki et al., Biochem. Biophys. Res. Commun, 244, 210 -219). Taking advantage of this reaction, we developed a highly sensit ive method for strand-specific detection of DNA or RNA. The probe cons isted of a 40- to 50-base-long complementary sequence on the 5' side a nd 10 repeats of AT on the 3' side. After hybridization using the 5' s ide, the 3' side AT repeat region was elongated by Delta Tth DNA polym erase in the presence of the dATP, dTTP, and digoxigenin (dig)-11-dUTP . The elongation condition was 52-62 degrees C for 3 h, The method nam ed HybrAT (hybridization-AT-tailing) was at least 100-fold more sensit ive than the conventional hybridization with 5' end dig-11-dUTP labele d probe. (C) 1998 Academic Press.