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Phytoplasma pp 395-403 | Cite as

Pulsed-Field Gel Electrophoresis for Isolation of Full-Length Phytoplasma Chromosomes from Plants

  • Carmine MarconeEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 938)

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.

Key words

Agarose blocks Catharanthus roseus CHEF system Full-length phytoplasma chromosome Phloem preparation Pulsed-field gel electrophoresis 

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Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  1. 1.Department of Pharmaceutical and Biomedical SciencesUniversity of SalernoSalernoItaly

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