Methods for cDNA cloning and sequencing tobacco mosaic virus RNA

Philip Goelet, Jonathan Karn

Research output: Contribution to journalArticlepeer-review

Abstract

DNA complementary to tobacco mosaic virus (TMV) RNA (cDNA) was prepared by priming reverse transcription with synthetic oligonucleotides. The cDNAs terminated prematurely at many specific sites and no transcripts longer than about 2000 nucleotides were obtained. However, the entire 6395 nucleotide long TMV RNA could be copied into cDNA by specific priming with a series of 13 to 17 residue long oligonucleotides or by non-specific priming with short, 4 to 7 residue, oligonucleotides. A number of different priming methods were used to convert the cDNA into double-stranded DNA (dsDNA). The double-stranded cDNA was recovered by shotgun cloning into M13 and analysed by sequencing. The frequency at which cDNA clones were recovered has been used to compare various cDNA cloning strategies.

Original languageEnglish (US)
Pages (from-to)331-342
Number of pages12
JournalGene
Volume29
Issue number3
DOIs
StatePublished - Sep 1984
Externally publishedYes

Keywords

  • M13 phage vector
  • Recombinant DNA bank
  • reverse transcription
  • second strand DNA synthesis
  • synthetic oligonucleotides

ASJC Scopus subject areas

  • Genetics

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