A protocol for the rapid isolation of full geminivirus genomes from dried plant tissue

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dc.contributor.authorShepherd, Dionne N
dc.contributor.authorMartin, Darren P
dc.contributor.authorLefeuvre, Pierre
dc.contributor.authorMonjane, Aderito L
dc.contributor.authorOwor, Betty E
dc.contributor.authorRybicki, Edward P
dc.contributor.authorVarsani, Arvind
dc.date.accessioned2016-07-27T12:01:49Z
dc.date.available2016-07-27T12:01:49Z
dc.date.issued2008
dc.date.updated2016-07-12T16:06:05Z
dc.description.abstractA high-throughput method of isolating and cloning geminivirus genomes from dried plant material, by combining an Extract-n-AmpTM-based DNA isolation technique with rolling circle amplification (RCA) of viral DNA, is presented. Using this method an attempt was made to isolate and clone full geminivirus genomes/genome components from 102 plant samples, including dried leaves stored at room temperature for between 6 months and 10 years, with an average hands-on-time to RCA-ready DNA of 15 min per 20 samples. While storage of dried leaves for up to 6 months did not appreciably decrease cloning success rates relative to those achieved with fresh samples, efficiency of the method decreased with increasing storage time. However, it was still possible to clone virus genomes from 47% of 10-year-old samples. To illustrate the utility of this simple method for high-throughput geminivirus diversity studies, six Maize streak virus genomes, an Abutilon mosaic virus DNA-B component and the DNA-A component of a previously unidentified New Word begomovirus species were fully sequenced. Genomic clones of the 69 other viruses were verified as such by end sequencing. This method should be extremely useful for the study of any circular DNA plant viruses with genome component lengths smaller than the maximum size amplifiable by RCA.en_ZA
dc.identifierhttp://dx.doi.org/10.1016/j.jviromet.2007.12.014
dc.identifier.apacitationShepherd, D. N., Martin, D. P., Lefeuvre, P., Monjane, A. L., Owor, B. E., Rybicki, E. P., & Varsani, A. (2008). A protocol for the rapid isolation of full geminivirus genomes from dried plant tissue. <i>Journal of Virological Methods</i>, http://hdl.handle.net/11427/20887en_ZA
dc.identifier.chicagocitationShepherd, Dionne N, Darren P Martin, Pierre Lefeuvre, Aderito L Monjane, Betty E Owor, Edward P Rybicki, and Arvind Varsani "A protocol for the rapid isolation of full geminivirus genomes from dried plant tissue." <i>Journal of Virological Methods</i> (2008) http://hdl.handle.net/11427/20887en_ZA
dc.identifier.citationShepherd, D. N., Martin, D. P., Lefeuvre, P., Monjane, A. L., Owor, B. E., Rybicki, E. P., & Varsani, A. (2008). A protocol for the rapid isolation of full geminivirus genomes from dried plant tissue. Journal of virological methods, 149(1), 97-102.en_ZA
dc.identifier.issn0166-0934en_ZA
dc.identifier.ris TY - Journal Article AU - Shepherd, Dionne N AU - Martin, Darren P AU - Lefeuvre, Pierre AU - Monjane, Aderito L AU - Owor, Betty E AU - Rybicki, Edward P AU - Varsani, Arvind AB - A high-throughput method of isolating and cloning geminivirus genomes from dried plant material, by combining an Extract-n-AmpTM-based DNA isolation technique with rolling circle amplification (RCA) of viral DNA, is presented. Using this method an attempt was made to isolate and clone full geminivirus genomes/genome components from 102 plant samples, including dried leaves stored at room temperature for between 6 months and 10 years, with an average hands-on-time to RCA-ready DNA of 15 min per 20 samples. While storage of dried leaves for up to 6 months did not appreciably decrease cloning success rates relative to those achieved with fresh samples, efficiency of the method decreased with increasing storage time. However, it was still possible to clone virus genomes from 47% of 10-year-old samples. To illustrate the utility of this simple method for high-throughput geminivirus diversity studies, six Maize streak virus genomes, an Abutilon mosaic virus DNA-B component and the DNA-A component of a previously unidentified New Word begomovirus species were fully sequenced. Genomic clones of the 69 other viruses were verified as such by end sequencing. This method should be extremely useful for the study of any circular DNA plant viruses with genome component lengths smaller than the maximum size amplifiable by RCA. DA - 2008 DB - OpenUCT DP - University of Cape Town J1 - Journal of Virological Methods LK - https://open.uct.ac.za PB - University of Cape Town PY - 2008 SM - 0166-0934 T1 - A protocol for the rapid isolation of full geminivirus genomes from dried plant tissue TI - A protocol for the rapid isolation of full geminivirus genomes from dried plant tissue UR - http://hdl.handle.net/11427/20887 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/20887
dc.identifier.vancouvercitationShepherd DN, Martin DP, Lefeuvre P, Monjane AL, Owor BE, Rybicki EP, et al. A protocol for the rapid isolation of full geminivirus genomes from dried plant tissue. Journal of Virological Methods. 2008; http://hdl.handle.net/11427/20887.en_ZA
dc.languageengen_ZA
dc.publisherElsevieren_ZA
dc.publisher.institutionUniversity of Cape Town
dc.rightsCreative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)*
dc.rights.urihttps://creativecommons.org/licenses/by-nc-nd/4.0/.en_ZA
dc.sourceJournal of Virological Methodsen_ZA
dc.source.urihttp://www.sciencedirect.com/science/journal/01660934
dc.subject.otherMaize streak virus (MSV)
dc.subject.otherGeminivirus
dc.subject.otherExtract-n-Amp
dc.subject.otherRolling circle amplification
dc.subject.otherGeminivirus dive
dc.titleA protocol for the rapid isolation of full geminivirus genomes from dried plant tissueen_ZA
dc.typeJournal Articleen_ZA
uct.type.filetype
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceArticleen_ZA
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