title: Sequencing of the large dsDNA genome of Oryctes rhinoceros nudivirus using multiple displacement amplification of nanogram amounts of virus DNA.
abstract: 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 phi29 DNA polymerase- and exonuclease-resistant random hexamer-based multiple displacement amplification (MDA) method. About 60 microg 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.
authors.affiliation: Laboratory for Biotechnological Crop Protection, Department of Phytopathology, Agricultural Service Center Palatinate (DLR Rheinpfalz), Breitenweg 71, 67435, Neustadt an der Weinstrasse, Germany.
authors.forename: Yongjie
authors.initials: Y
authors.lastname: Wang
authors.forename: Regina G
authors.initials: RG
authors.lastname: Kleespies
authors.forename: Moslim B
authors.initials: MB
authors.lastname: Ramle
authors.forename: Johannes A
authors.initials: JA
authors.lastname: Jehle
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000145:classification / Q000235:genetics*; D001517:Coleoptera / Q000821:virology; D004247:DNA / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D016679:Genome, Viral / Q000235:genetics*; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015183:Restriction Mapping
chemical_list: D004279:DNA, Viral; D004247:DNA