title: Pulsed-field gel electrophoresis for isolation of full-length phytoplasma chromosomes from plants.
abstract: Pulsed-field gel electrophoresis (PFGE) is a powerful technique for genomic studies of unculturable plant-pathogenic phytoplasmas, which enables separation of full-length phytoplasma chromosomes from contaminating host plant nucleic acids. The PFGE method described here involves isolation of phytoplasmal DNA from high-titer phytoplasma-infected herbaceous plants using a phytoplasma enrichment procedure, embedding of phytoplasma chromosomes in agarose blocks, and separation of entire phytoplasma chromosomes from contaminating host plant nucleic acids by electrophoresis. Full-length phytoplasma chromosomes are resolved as single, discrete bands in the gel. The identity of these bands can be confirmed by Southern blot hybridization using a ribosomal DNA fragment as a probe. The method does not utilize gamma-irradiation to linearize phytoplasma chromosomes prior to electrophoresis.
authors.affiliation: Department of Pharmaceutical and Biomedical Sciences, University of Salerno, Salerno, Italy. cmarcone@unisa.it
authors.forename: Carmine
authors.initials: C
authors.lastname: Marcone
mesh_terms: D030008:Catharanthus / Q000235:genetics / Q000382:microbiology; D002876:Chromosomes, Bacterial; D004269:DNA, Bacterial / Q000302:isolation & purification*; D018744:DNA, Plant / Q000302:isolation & purification*; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods; D045582:Phytoplasma / Q000235:genetics*; D010944:Plants / Q000235:genetics / Q000382:microbiology*
chemical_list: D004269:DNA, Bacterial; D018744:DNA, Plant