Reverse transcription polymerase chain reaction protocols for cloning small circular RNAs

J Virol Methods. 1998 Jul;73(1):1-9. doi: 10.1016/s0166-0934(98)00042-1.

Abstract

A protocol is described for general application for cloning small circular RNAs which requires only minimal amounts of template (approximately 50 ng) of unknown sequence. Both cDNA strands are synthesized with a 26-mer primer whose six 3'-terminal positions are totally degenerate in two consecutive reactions catalyzed by reverse transcriptase and DNA polymerase, respectively. The cDNAs are then PCR-amplified, using a 20-mer primer with the non-degenerate sequence of the previous primer, cloned and sequenced. This information permits the synthesis of one or more pairs of specific and adjacent primers for obtaining full-length cDNA clones by a protocol which is also described.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Southern
  • Cloning, Molecular / methods*
  • DNA Polymerase I
  • DNA Primers
  • DNA, Complementary / biosynthesis
  • DNA, Complementary / genetics
  • In Situ Hybridization
  • Molecular Weight
  • Nucleic Acid Denaturation
  • Polymerase Chain Reaction / methods*
  • RNA / genetics*
  • RNA / isolation & purification
  • RNA, Circular
  • RNA, Satellite / genetics
  • RNA, Viral / genetics
  • RNA-Directed DNA Polymerase
  • Sequence Analysis, DNA
  • Viroids / genetics

Substances

  • DNA Primers
  • DNA, Complementary
  • RNA, Circular
  • RNA, Satellite
  • RNA, Viral
  • RNA
  • RNA-Directed DNA Polymerase
  • DNA Polymerase I