Direct Sequencing by Thermal Asymmetric PCR

Abstract

Direct sequencing of PCR products (1) has proven to be a powerful method in the generation of nucleic acid sequence data. Using these techniques, it is possible to produce microgram quantities of pure target DNA and subsequently its nucleotide sequence in a few hours, even, theoretically, from one single RNA or DNA molecule. However, problems have been encountered, and these have been attributed to the strong tendency of the short double-strand DNA templates to reanneal. In fact, compared to double-stranded plasmid DNA, which can be permanently denatured by alkali treatment and then form intermolecular interactions compatible with good sequencing efficiency, optimized conditions for direct sequencing are required before reannealing with short PCR product.