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Rapid and Simple Amplification of Genomic DNA Sequences Flanking Transposon

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Author(s): ARIS TRI WAHYUDI

Journal: Microbiology Indonesia
ISSN 1978-3477

Volume: 1;
Issue: 1;
Start page: 1;
Date: 2007;
Original page

Keywords: inverse PCR | Tansposon Mini-Tn5Km1 | Southern Hybridization | DNA sequencing

ABSTRACT
A rapid and simple method to amplify genomic DNA sequences flanking mini-Tn5 transposon insertion wasdeveloped. This technique can be used to determine the location and orientation of the transposon insertion within genomic DNA of the bacteria. Based on the mini-Tn5Km1 transposon sequence, PCR primers can be designed to specifically amplify the DNA sequences flanking mini-Tn5 transposon by inverse polymerase chain reaction (inverse PCR) directly, upstream and downstream of the transposon insertion. The method involves: (i) digestion with a restriction enzyme that does not cut mini-Tn5Km1 sequence; (ii) self-ligation under conditions favoring the production of monomeric circles; and (iii) inverse PCR reaction using primers designed from mini-Tn5Km1 sequence to amplify the DNA sequences flanking mini-Tn5Km1 transposon insertion. Feasibility and reliability of this method were demonstrated with mini-Tn5Km1 mutants of the microaerobic magnetic bacterium Magnetospirillum magneticum AMB-1 which are defective in magnetosomes synthesis. The inverse PCR products amplified from these mutant genomes showed the correct fragments as determined through Southern hybridization and DNA sequence analysis.
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