TY - JOUR
T1 - A protocol for the rapid isolation of full geminivirus genomes from dried plant tissue
AU - Shepherd, Dionne N.
AU - Martin, Darren P.
AU - Lefeuvre, Pierre
AU - Monjane, Adérito L.
AU - Owor, Betty E.
AU - Rybicki, Edward P.
AU - Varsani, Arvind
N1 - Funding Information:
This research was partially funded by the National Research Foundation (South Africa). DNS is supported by PANNAR (Pty) Ltd.; DPM is supported by the Wellcome Trust, the Harry Oppenheimer Trust and the Sydney Brenner Fellowship; PL is supported by CIRAD and the French Ministère de la Recherche et de l’Enseignement Supérieur; BO is supported by the Rockefeller foundation through the USHEPiA programme; ALM is supported by the Canon Collins Trust for Southern Africa and a University of Cape Town International Scholarship; AV is supported by the Carnegie Corporation of New York.
PY - 2008/4
Y1 - 2008/4
N2 - A high-throughput method of isolating and cloning geminivirus genomes from dried plant material, by combining an Extract-n-Amp™-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.
AB - A high-throughput method of isolating and cloning geminivirus genomes from dried plant material, by combining an Extract-n-Amp™-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.
KW - Extract-n-Amp
KW - Geminivirus
KW - Geminivirus diversity
KW - Large scale plant sampling
KW - Maize streak virus (MSV)
KW - Rapid virus DNA extraction
KW - Rolling circle amplification
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U2 - 10.1016/j.jviromet.2007.12.014
DO - 10.1016/j.jviromet.2007.12.014
M3 - Article
C2 - 18280590
AN - SCOPUS:40849083670
SN - 0166-0934
VL - 149
SP - 97
EP - 102
JO - Journal of Virological Methods
JF - Journal of Virological Methods
IS - 1
ER -