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

J Virol Methods. 2008 Sep;152(1-2):106-8. doi: 10.1016/j.jviromet.2008.06.003. Epub 2008 Jul 18.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Baculoviridae / classification
  • Baculoviridae / genetics*
  • Coleoptera / virology
  • DNA / genetics*
  • DNA, Viral / genetics*
  • Genome, Viral / genetics*
  • Molecular Sequence Data
  • Nucleic Acid Amplification Techniques / methods*
  • Restriction Mapping

Substances

  • DNA, Viral
  • DNA

Associated data

  • GENBANK/EU747721