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Rapid identification and mapping of insertion sequences in Escherichia coli genomes using vectorette PCR

BioMed Central
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  • Methodology Article
  • Biology

Abstract ral ss BioMed CentBMC Microbiology Open AcceMethodology article Rapid identification and mapping of insertion sequences in Escherichia coli genomes using vectorette PCR Shaobin Zhong1 and Antony M Dean*1,2 Address: 1Biotechnology Institute, University of Minnesota, 1479 Gortner Ave., St. Paul, MN55108, USA and 2Department of Ecology, Evolution and Behavior, University of Minnesota, 1987 Upper Buford Circle, St. Paul, MN55108, USA Email: Shaobin Zhong - [email protected]; Antony M Dean* - [email protected] * Corresponding author Abstract Background: Insertion sequences (IS) are small DNA segments capable of transposing within and between prokaryotic genomes, often causing insertional mutations and chromosomal rearrangements. Although several methods are available for locating ISs in microbial genomes, they are either labor-intensive or inefficient. Here, we use vectorette PCR to identify and map the genomic positions of the eight insertion sequences (IS1, 2, 3, 4, 5, 30, 150, and 186) found in E. coli strain CGSC6300, a close relative of MG1655 whose genome has been sequenced. Results: Genomic DNA from strain CGSC6300 was digested with a four-base cutter Rsa I and the resulting restriction fragments ligated onto vectorette units. Using IS-specific primers directed outward from the extreme ends of each IS and a vectorette primer, flanking DNA fragments were amplified from all but one of the 37 IS elements identified in the genomic sequence of MG1655. Purification and sequencing of the PCR products confirmed that they are IS-associated flanking DNA fragments corresponding to the known IS locations in the MG1655 genome. Seven additional insertions were found in strain CGSC6300 indicating that very closely related isolates of the same laboratory strain (the K12 isolate) may differ in their IS complement. Two other E. coli K12 derivatives, TD2 and TD10, were also analyzed by vectorette PCR. They share 36 of the MG1655 IS sites as well as having 16 and 18 a

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