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Sequencing of the large dsDNA genome of Oryctes rhinoceros nudivirus using multiple displacement amplification of nanogram amounts of virus DNA

Zugehörigkeit
Laboratory for Biotechnological Crop Protection, Department of Phytopathology, Agricultural Service Center Palatinate (DLR Rheinpfalz), Neustadt an der Weinstrasse, Germany
Wang. Yongjie;
GND
1059093685
Zugehörigkeit
Julius Kühn-Institute (JKI), Institute for Biological Control, Germany
Kleespies, Regina G.;
Zugehörigkeit
Malaysian Palm Oil Board, Kuala Lumpur, Malaysia
Ramle, Moslim B.;
GND
17274184X
Zugehörigkeit
Laboratory for Biotechnological Crop Protection, Department of Phytopathology, Agricultural Service Center Palatinate (DLR Rheinpfalz), Neustadt an der Weinstrasse, Germany
Jehle, Johannes A.

The genomic sequence analysis of many large dsDNA viruses is hampered by the lack of enough sample materials. Here,we report a whole genomeamplification of the Oryctes rhinoceros nudivirus (OrNV) isolate Ma07 starting from as few as about 10 ng of purified viral DNA by application of 29 DNA polymeraseand exonuclease-resistant random hexamer-based multiple displacement amplification (MDA) method. About 60g of high molecular weight DNA with fragment sizes of up to 25 kbp was amplified. A genomic DNA clone librarywas generated using the product DNA. After 8-fold sequencing coverage, the 127,615 bp of OrNV whole genome was sequenced successfully. The results demonstrate that the MDA-based whole genome amplification enables rapid access to genomic information from exiguous virus samples.

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