title: Mapping Transposon Insertions in Bacterial Genomes by Arbitrarily Primed PCR.
abstract: Transposons can be used to easily generate and label the location of mutations throughout bacterial and other genomes. Transposon insertion mutants may be screened for a phenotype as individual isolates, or by selection applied to a pool of thousands of mutants. Identifying the location of a transposon insertion is critical for connecting phenotype to the genetic lesion. In this unit, we present an easy and detailed approach for mapping transposon insertion sites using arbitrarily-primed PCR (AP-PCR). Two rounds of PCR are used to (1) amplify DNA spanning the transposon insertion junction, and (2) increase the specific yield of transposon insertion junction fragments for sequence analysis. The resulting sequence is mapped to a bacterial genome to identify the site of transposon insertion. In this protocol, AP-PCR as it is routinely used to map sites of transposon insertion within Staphylococcus aureus, is used to illustrate the principle. Guidelines are provided for adapting this protocol for mapping insertions in other bacterial genomes. Mapping transposon insertions using this method is typically achieved in 2 to 3 days if starting from a culture of the transposon insertion mutant. © 2017 by John Wiley & Sons, Inc.
authors.affiliation: Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts.
authors.forename: José T
authors.initials: JT
authors.lastname: Saavedra
authors.affiliation: Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts.|Department of Ophthalmology, and Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts.|The Broad Institute, Cambridge, Massachusetts.
authors.forename: Julia A
authors.initials: JA
authors.lastname: Schwartzman
authors.affiliation: Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts.|Department of Ophthalmology, and Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts.|The Broad Institute, Cambridge, Massachusetts.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Gilmore
mesh_terms: D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods*; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics*; D016680:Genome, Bacterial; D016133:Polymerase Chain Reaction / Q000379:methods*; D013211:Staphylococcus aureus / Q000235:genetics*
chemical_list: D004251:DNA Transposable Elements; D004269:DNA, Bacterial
keywords: anchored PCR; arbitrarily primed PCR; gene-walking PCR; transposon