title: Preparation of viral DNA from nucleocapsids.
abstract: Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.(1,2,3) This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.(4,5) The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD(260/280;) of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 μg or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 10(6) PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 10(8) plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.(6) For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 μl of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate). Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.
authors.affiliation: Department of Molecular Biology, Princeton University.
authors.forename: Moriah L
authors.initials: ML
authors.lastname: Szpara
authors.forename: Yolanda R
authors.initials: YR
authors.lastname: Tafuri
authors.forename: L W
authors.initials: LW
authors.lastname: Enquist
mesh_terms: D000818:Animals; D002499:Centrifugation, Density Gradient / Q000379:methods; D002522:Chlorocebus aethiops; D004279:DNA, Viral / Q000302:isolation & purification*; D019251:Nucleocapsid / Q000737:chemistry / Q000235:genetics*; D014709:Vero Cells
chemical_list: D004279:DNA, Viral