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

• Published in: Journal of virological methods 2008-09, Vol.152 (1), p.106-108
• Main Authors: Wang, Yongjie, Kleespies, Regina G., Ramle, Moslim B., Jehle, Johannes A.
• Format: Article
• Language: English
• Subjects: Animals; Baculoviridae - classification; Baculoviridae - genetics; Biological and medical sciences; Coleoptera - virology; DNA - genetics; DNA, Viral - genetics; Fundamental and applied biological sciences. Psychology; Genome sequencing; Genome, Viral - genetics; Large dsDNA viruses; Microbiology; Molecular Sequence Data; Nucleic Acid Amplification Techniques - methods; Oryctes rhinoceros; Oryctes rhinoceros nudivirus (OrNV); Restriction Mapping; Techniques used in virology; Virology; Whole genome amplification
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2008.06.003

The genomic sequence analysis of many large dsDNA viruses is hampered by the lack of enough sample materials. Here, we report a whole genome amplification of the Oryctes rhinoceros nudivirus (OrNV) isolate Ma07 starting from as few as about 10 ng of purified viral DNA by application of ϕ29 DNA polymerase- and exonuclease-resistant random hexamer-based multiple displacement amplification (MDA) method. About 60 μg of high molecular weight DNA with fragment sizes of up to 25 kbp was amplified. A genomic DNA clone library was 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.