Dideoxy Sequencing of RNA Using Reverse Transcriptase

Abstract

Shortly after the dideoxy (dd) chain-termination method for DNA sequencing was described by Sanger et al., Zimmern and Kaesberg modified this method to sequence RNA directly, using purified encephalomyocarditis viral RNA as a template, pdT_(7)rC as a specific primer, and reverse transcriptase as the elongation enzyme. Bina-Stein et al. further developed the method to use total cellular mRNA as a template and complementary DNA restriction fragments, which anneal to a particular mRNA at specific locations, as primers for reverse transcriptase. Recently, sequence-specific oligonucleotides, synthesized with an automated DNA synthesizer, have been used as primers on both DNA and RNA templates.

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