Efficient amplification of multiple transposon-flanking sequences

J Microbiol Methods. 2000 Aug;41(3):195-9. doi: 10.1016/s0167-7012(00)00159-7.

Abstract

Transposon mutagenesis is a very useful tool for gene identification in bacteria. Once the transposon mutants of interest are isolated, it is often necessary to identify the sequences that flank the transposon insertions. We devised an efficient method for specific amplification of transposon-flanking sequences that requires the sequence information of only transposon-specific sequences. The basic steps for this method consists of (1) digestion with a restriction enzyme, (2) ligation with a Y-shaped linker and (3) polymerase chain reaction amplification using a transposon-specific primer and a primer specific to the Y-shaped linker. The feasibility of this method was demonstrated with mini-Tn5 mutants of Salmonella typhimurium. We also found that this method can be used for simultaneous amplification of multiple transposon-flanking sequences.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions / genetics
  • 3' Untranslated Regions / metabolism
  • 5' Untranslated Regions / genetics
  • 5' Untranslated Regions / metabolism
  • DNA Transposable Elements / genetics*
  • DNA, Bacterial / chemistry
  • Electrophoresis, Agar Gel
  • Mutagenesis, Insertional / methods
  • Nucleic Acid Amplification Techniques*
  • Oligonucleotide Probes / chemical synthesis
  • Polymerase Chain Reaction
  • Salmonella typhimurium / genetics
  • Templates, Genetic

Substances

  • 3' Untranslated Regions
  • 5' Untranslated Regions
  • DNA Transposable Elements
  • DNA, Bacterial
  • Oligonucleotide Probes