Novel method of detecting single base substitutions in RNA molecules by differential melting behavior in solution

Genomics. 1988 Oct;3(3):217-23. doi: 10.1016/0888-7543(88)90082-1.

Abstract

We have developed a quick and reliable way of detecting point mutations in RNA molecules. This method involves melting RNA-RNA heteroduplexes of varying lengths in a series of tubes containing a stepwise salt or formamide gradient, followed by polyacrylamide gel electrophoresis to distinguish between single- and double-stranded RNA molecules. The manipulations required are technically simple, and the method is sensitive enough to detect destabilization of the highest melting domain of a dsRNA duplex by a single base mismatch. When this method is used in parallel with denaturing gradient gel electrophoresis, which detects point mutations in low-melting domains of duplexes, it should now be possible to rapidly screen for mutations located throughout the length of any RNA molecule whose wild-type sequence is known.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Composition
  • Cloning, Molecular
  • DNA, Viral / genetics
  • Electrophoresis, Polyacrylamide Gel
  • Genes, Viral*
  • Influenza A virus / genetics*
  • Mutation*
  • Nucleic Acid Denaturation
  • Nucleic Acid Hybridization
  • Plasmids
  • RNA, Viral / genetics*
  • Transcription, Genetic

Substances

  • DNA, Viral
  • RNA, Viral