Bidirectional chain-termination nucleotide sequencing: transposon Tn5seq1 as a mobile source of primer sites

Dilip K. Nag, Henry V. Huang, Douglas E. Berg

Research output: Contribution to journalArticlepeer-review

36 Scopus citations

Abstract

The sequencing of large DNA fragments by the chain-termination method [Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467] has generally required extensive manipulations to bring all parts of the fragment near a specific primer-binding site, or the repeated synthesis of new oligodeoxynucleotide primers. Here we develop a more efficient approach, the use of a transposable element to insert primer binding sites at random in the DNA of interest. We constructed a Tn5 derivative called Tn5 seql with unique DNA segments near each end so that oligodeoxynucleotides matching them could serve as primers for sequencing in each direction from any Tn5seql insertion site. Our experiments demonstrate the use of Tn5 seql for sequencing in pBR322 plasmids and also in uncloned DNAs of the Escherichia coli chromosome. The unique segments near the left and right ends of Tn5 seql are promoters from phages T7 and SP6, respectively, to permit the efficient transcription of adjacent DNAs in vivo or in vitro.

Original languageEnglish
Pages (from-to)135-145
Number of pages11
JournalGene
Volume64
Issue number1
DOIs
StatePublished - Apr 15 1988

Keywords

  • Recombinant DNA
  • bacteriophages λ, T7, SP6
  • insertion mutagenesis
  • promoters
  • transcription

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