RNA sequencing by primer extension

Direct sequencing of RNA by primer extension is a fast and accurate method that is useful in a variety of situations. It is a valuable technique for determining the faithfulness of in vitro processing reactions, such as splicing or RNA editing. It is often used as an alternative to reverse transcription PCR (RT-PCR) followed by cloning and DNA sequencing. In the primer extension reaction, a radiolabeled probe (almost always a 5′-end-labeled DNA oligonucleotide) is annealed to the target RNA of interest. After hybridization, cDNA synthesis by reverse transcription proceeds from the 3′ end of the primer in the presence of chain-terminating dideoxynucleotide triphosphates. The cDNA products are fractionated on denaturing polyacrylamide gels and analyzed by phosphorimaging or autoradiography.

Nilsen TW. (2013) RNA sequencing by primer extension. Cold Spring Harb Protoc 2013(12). [abstract]