Sensitive detection of RNA using strand-specific M13 probes

Daniel M. Brown, Jonathan Frampton, Philip Goelet, Jonathan Kam

Research output: Contribution to journalArticle

Abstract

We have extended the method of Hu and Messing (Gene 17 (1982) 271-277) to prepare highly radioactive M13 probes suitable for use in RNA-DNA hybridization experiments. Single strands of M13 DNA carrying cloned sequences are rendered partially double-stranded by primed synthesis using a synthetic oligonucleotide primer complementary to a region 5′ to the cloning site. The newly synthesized radioactive complementary strand is then covalently cross-linked to the M13 phage DNA by UV irradiation in the presence of 4,5,8-trimethylpsoralen (trioxsalen). Since the cross-linked probe is stable to heat denaturation, and the region of cloned sequence is kept single-stranded, these complexes may be used as strand-specific hybridization probes to detect RNA sequences under conditions which would denature DNA-DNA duplexes.

Original languageEnglish (US)
Pages (from-to)139-144
Number of pages6
JournalGene
Volume20
Issue number2
DOIs
StatePublished - Dec 1982
Externally publishedYes

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Keywords

  • Northern blotting
  • Oligonucleotide primer
  • plaque hybridization
  • trioxsalen crosslinking

ASJC Scopus subject areas

  • Genetics

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