title: Locating transposable element polymorphisms in bacterial genomes.
abstract: Although whole-genome sequencing is greatly extending our knowledge of the genetic capacity of those bacterial species, it is only directly informative for the particular strain sequenced. Many bacterial species exhibit more or less genetic polymorphism within their populations and characterising this variety is an extremely important way of elucidating the biology of these species. Often genomic polymorphisms are associated with multicopy elements, particularly transposable elements. We describe a novel method that efficiently characterises the sequences of such polymorphisms. We have optimised heminested inverse PCR (hINVPCR) to assess the diversity of insertional polymorphisms of a transposable element (IS6110) in clinical isolates of Mycobacterium tuberculosis. To increase the yield of information, genomic DNA was digested with different endonucleases (Bsp1286I, HaeII or PvuI), and primers based on both the 5' and 3' ends of IS6110 were used to amplify and determine the genomic sequence upstream (or downstream) of the transposable element. We found that both the choice of restriction enzyme and the use of primers at both ends of the transposable element significantly increased the diversity of the insertion sites identified. Band stabbing was incorporated into the method as an alternative to cloning in order to screen large number of isolates at a sequence level in a rapid and labour-efficient fashion. We describe some of the purposes to which such data can be put.
authors.affiliation: Department of Medical Microbiology, University of Aberdeen, Medical School Building, Foresterhill, UK. hy@abdn.ac.uk
authors.forename: Hasan
authors.initials: H
authors.lastname: Yesilkaya
authors.forename: Anne
authors.initials: A
authors.lastname: Thomson
authors.forename: Christine
authors.initials: C
authors.lastname: Doig
authors.forename: Brian
authors.initials: B
authors.lastname: Watt
authors.forename: Jeremy W
authors.initials: JW
authors.lastname: Dale
authors.forename: Ken J
authors.initials: KJ
authors.lastname: Forbes
mesh_terms: D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis; D016680:Genome, Bacterial; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D004251:DNA Transposable Elements; D004269:DNA, Bacterial