Application of DNA Topoisomerase-Activated Adapters to Riboprobe Synthesis

Abstract

Topoisomerase-activated adapters for rapid incorporation of the T7 promoter into PCR products were made by hybridizing synthetic oligonucleotides and activating vaccinia DNA topoisomerase I. The adapters were used to incorporate the T7 promoter sequence into PCR products amplified from cDNA and genomic DNA. Modified PCR products were used as templates to synthesize digoxigenin-labeled sense and cRNA probes by in vitro transcription with phage T7 RNA polymerase. The red/green cones were labeled by the antisense probe, but no specific signal was produced by the sense probe. These results demonstrate that topoisomerase-activated adapters provide a powerful and convenient tool for the rapid modification of PCR products.