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Matus Valach 2016. Primer extension assay and sequencing by dideoxynucleotide incorporation on RNA templates. protocols.io https://dx.doi.org/10.17504/protocols.io.ejtbcnnCopy Citation Copied
URL: https://dx.doi.org/DOI:10.17504/protocols.io.ejtbcnn
Authors: Matus Valach
Summary: The identity of a nucleotide or the presence of a bulky modification or strand break in an RNA can be determined by several approaches. When the 3′ region of the analyzed RNA is known, extending a (radio)labeled primer by reverse transcription and analyzing the reaction products using denaturing polyacrylamide gel electrophoresis and subsequent (radio)imaging allows mapping of cDNA chain-termination sites. (Based on the design of the experiment, these can be, for example, a strand break, 5′ end, pause-inducing site, or site of dideoxynucleotide incorporation.) This procedure is adapted from the previously published protocol by Walker and Lorsch (DOI: 10.1016/B978-0-12-420037-1.00020-8).
Affiliations: Université de Montréal, Montreal, Quebec, Canada
Version: 1
Publication Date: 2016
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