An alternative inverse PCR (IPCR) method to amplify DNA sequences flanking Tn5 transposon insertions.
We have developed an alternative method to amplify DNA sequences flanking Tn5 transposon insertions. This method relies on the identical sequences of inverted terminal repeats, located at the 5' and 3' ends of Tn5, to determine the location and orientation of a transposon insertion within a restriction endonuclease fragment. From this information, PCR primers can be designed to selectively amplify by inverse PCR the DNA flanking one side of the transposon. This method avoids the problem of amplifying or cloning long sequences flanking Tn5. To demonstrate the applicability of this method, we generated Tn5 transposon mutants of Pseudomonas abietaniphila BKME-9 which no longer grew on dehydroabietic acid (DhA). The flanking sequence of one of the mutant (strain BKME-941) which accumulated 7-oxoDhA, was amplified.
Martin
V J
VJ
Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada.
Mohn
W W
WW
D045784:Abietanes; D015139:Blotting, Southern; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D004224:Diterpenes / Q000378:metabolism; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction / Q000379:methods*; D011549:Pseudomonas / Q000235:genetics* / Q000254:growth & development
D045784:Abietanes; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004224:Diterpenes; C013913:dehydroabietic acid