Rapid and Simple Amplification of Genomic DNA Sequences Flanking Transposon  

Oleh:

Wahyudi, Aris Tri

Jenis: Article from Journal - ilmiah nasional - terakreditasi DIKTI
Dalam koleksi: Microbiology Indonesia vol. 01 no. 01 (Apr. 2007), page 1-4.
Topik: Inverse PCR; Transposon Mini-Tn5Kml; Southern Hybridization; DNA Sequencing
Fulltext: Aris Tri Wahyudi.pdf (1,011.67KB)

Ketersediaan

Perpustakaan Pusat (Semanggi) Nomor Panggil: MM78.1 Non-tandon: 1 (dapat dipinjam: 0) Tandon: tidak ada	Lihat Detail Induk
Perpustakaan FK Nomor Panggil: M53.K Non-tandon: 1 (dapat dipinjam: 0) Tandon: tidak ada	Lihat Detail Induk

Isi artikel

A rapid and simple method to amplify genomic DNA sequences flanking mini-Tn5 transposon insertion was developed. 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-Tn5Kml 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-Tn5Kml sequence; (ii) self-ligation under conditions favoring the production of monomeric circles; and (iii) inverse PCR reaction using primers designed from mini- Tn5Kml sequence to amplify the DNA sequences flanking mini-Tn5Kml transposon insertion. Feasibility and reliability of this method were demonstrated with mini-Tn5Kml 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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