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In-Gel Isolation and Characterization of Large (and Other) Phages

Department of Biochemistry and Structural Biology, The University of Texas Health Center, San Antonio, TX 78229-3900, USA
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Viruses 2020, 12(4), 410; https://doi.org/10.3390/v12040410
Received: 11 March 2020 / Revised: 2 April 2020 / Accepted: 5 April 2020 / Published: 7 April 2020
(This article belongs to the Special Issue Giant or Jumbo Phages)
We review some aspects of the rapid isolation of, screening for and characterization of jumbo phages, i.e., phages that have dsDNA genomes longer than 200 Kb. The first aspect is that, as plaque-supporting gels become more concentrated, jumbo phage plaques become smaller. Dilute agarose gels are better than conventional agar gels for supporting plaques of both jumbo phages and, prospectively, the even larger (>520 Kb genome), not-yet-isolated mega-phages. Second, dilute agarose gels stimulate propagation of at least some jumbo phages. Third, in-plaque techniques exist for screening for both phage aggregation and high-in-magnitude, negative average electrical surface charge density. The latter is possibly correlated with high phage persistence in blood. Fourth, electron microscopy of a thin section of a phage plaque reveals phage type, size and some phage life cycle information. Fifth, in-gel propagation is an effective preparative technique for at least some jumbo phages. Sixth, centrifugation through sucrose density gradients is a relatively non-destructive jumbo phage purification technique. These basics have ramifications in the development of procedures for (1) use of jumbo phages for phage therapy of infectious disease, (2) exploration of genomic diversity and evolution and (3) obtaining accurate metagenomic analyses. View Full-Text
Keywords: agarose; electron microscopy; genomic DNA sequencing; in-plaque analysis; phage therapy of infectious disease; rate zonal centrifugation in sucrose gradients agarose; electron microscopy; genomic DNA sequencing; in-plaque analysis; phage therapy of infectious disease; rate zonal centrifugation in sucrose gradients
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Serwer, P.; Wright, E.T. In-Gel Isolation and Characterization of Large (and Other) Phages. Viruses 2020, 12, 410.

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