Submitted to: Journal of Virological Methods
Publication Type: Peer reviewed journal
Publication Acceptance Date: 11/27/2008
Publication Date: 1/22/2009
Citation: Volkening, J.D., Spatz, S.J. 2009. Purification of DNA from the cell-associated herpesvirus Marek's disease virus for 454 pyrosequencing using micrococcal nuclease digestion and polyethylene glycol precipitation. Journal of Virological Methods. 157(1):55-61. Interpretive Summary: To aid in our comparative genomic efforts in understanding the genetic basis of GaHV-2 virulence we have investigated various methods for the isolation of viral DNA suitable for 454 pyrosequencing, a technology that does not rely on ‘Shot-gun’ cloning of fragmented DNA. The ultimate goal was to develop a technique that would yield pristine GaHV-2 DNA with limited cellular DNA contamination, which does not rely on extensive serial propagation in cell culture, a process which has been shown to attenuate the virus. Various methodoloies were investigated and hypotonic lysis of infected cells, micrococcal exonuclease degradation of non–encapsulated DNA and removal of degraded DNA using polyethylene glycol precipitation results in the best conditions to yield suitable DNA.
Technical Abstract: Marek’s disease virus (MDV-1) is a cell-associated alphaherpesvirus that induces rapid-onset T-cell lymphomas in poultry. The genomes of 6 strains have been sequenced using both Sanger didoxy sequencing and 454 Life Science pyrosequencing. These genomes largely represent cell culture adapted strains due to the difficulty in obtaining large quantities of DNA from true low passage isolates. There are clear advantages from analyzing MDV-1 genomes directly from infected tissues or low passage isolates since serial passage attenuates the virus. We have attempted to use an ATP-dependent exonuclease and Phi29 DNA polymerase to selectively degrade host DNA and enzymatically amplify MDV-1 DNA from total DNA preps without much success. However, we have developed a protocol for purification of low passage MDV-1 DNA from infected avian fibroblasts. The method builds upon and extends available protocols based on hypotonic lysis to release virus particles followed by micrococcal nuclease treatment to degrade cellular DNA. Intact high molecular weight viral DNA is purified away from an excess of degraded cellular DNA using polyethylene glycol precipitation. 454-based pyrosequencing of viral DNA purified in this manner has generated data containing as little as 2.3% host sequences. On average DNA preparations were 70% (+/- 20%) pure yielding a genome coverage range of 25 -74 fold.