DNA and RNA Sequence Determination Based on Phosphorothioate Chemistry

Abstract

The difference in reactivity between phosphate and phosphorothioate diesters is the basis of a chemical degradation scheme for the sequencing of DNA and RNA. The phosphorothioate groups are incorporated into the nucleic acid in four separate enzymatic reactions, with three of the natural nucleoside triphosphates and one alpha-thiotriphosphate in each reaction. Selective strand cleavage is achieved through alkylation to form the hydrolytically labile phosphorothioate triester. As an example, the sequence analysis is presented of M13 phage DNA and of RNA prepared by transcription with SP6 RNA polymerase.