Sequencing of the large dsDNA genome of Oryctes rhinoceros nudivirus using multiple displacement amplification of nanogram amounts of virus DNA.
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.
Wang
Yongjie
Y
Laboratory for Biotechnological Crop Protection, Department of Phytopathology, Agricultural Service Center Palatinate (DLR Rheinpfalz), Breitenweg 71, 67435, Neustadt an der Weinstrasse, Germany.
Kleespies
Regina G
RG
Ramle
Moslim B
MB
Jehle
Johannes A
JA
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
D004279:DNA, Viral; D004247:DNA