Rapid amplification and cloning of Tn5 flanking fragments by inverse PCR

Lett Appl Microbiol. 2000 Aug;31(2):149-53. doi: 10.1046/j.1365-2672.2000.00781.x.


A simple approach is described to efficiently amplify DNA sequences flanking transposon Tn5 insertions. The method involves: (i) digestion with a restriction enzyme that cuts within Tn5; (ii) self-ligation under conditions favouring the production of monomeric circles; (iii) four parallel PCR reactions using primers designed to amplify left or right flanking sequences, and to distinguish target amplicons from non-specific products. This reveals the number of Tn5 insertions and the size of flanking genomic restriction fragments, without Southern blot analysis. The amplified product contains restriction sites that facilitate cohesive-end cloning. This rapid method is demonstrated using Tn5 and Tn5-Mob tagged DNA sequences involved in albicidin biosynthesis in Xanthomonas albilineans. It is generally applicable for efficient recovery of DNA sequences flanking transposon Tn5 derivatives in insertional mutagenesis studies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / biosynthesis
  • Cloning, Molecular*
  • DNA Transposable Elements*
  • Deoxyribonuclease BamHI / metabolism
  • Gene Amplification*
  • Mutagenesis, Insertional
  • Organic Chemicals
  • Polymerase Chain Reaction / methods*
  • Sequence Analysis, DNA
  • Xanthomonas / genetics*


  • Anti-Bacterial Agents
  • DNA Transposable Elements
  • Organic Chemicals
  • albicidin
  • Deoxyribonuclease BamHI