Rapid amplification and cloning of Tn5 flanking fragments by inverse PCR.
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.
Huang
G
G
Department of Botany, University of Queensland, Brisbane, Australia.
Zhang
L
L
Birch
R G
RG
D000900:Anti-Bacterial Agents / Q000096:biosynthesis; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D015245:Deoxyribonuclease BamHI / Q000378:metabolism; D005784:Gene Amplification; D016254:Mutagenesis, Insertional; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D014974:Xanthomonas / Q000235:genetics*
D000900:Anti-Bacterial Agents; D004251:DNA Transposable Elements; D009930:Organic Chemicals; C046156:albicidin; D015245:Deoxyribonuclease BamHI