A simplified method for the extraction of baculoviral DNA for PCR analysis: a practical application.
There are two major strategies to genetically modify baculoviruses. One uses a bacmid-based system which replicates in Escherichia coli using a bacterial origin of replication. The other employs a transfer vector and viral DNA which are co-transfected into insect cells and utilise host enzyme-mediated homologous recombination. Putative recombinants are then typically screened by plaque assay. The bacmid system is more convenient, but it requires a number of complex construction and isolation steps to obtain the correct bacmid genome. Generally, the transfer vector method is preferable when only a small number of genetic modifications are required. In this study a rapid and reliable method was developed to extract baculovirus DNA for PCR analysis from cultured insect cells. Briefly, viral DNA was isolated in three steps: SDS lysis, chloroform extraction and ethanol precipitation. The method was tested for direct screening of recombinant viruses in plaque assays. Contrary to previous reports, baculovirus DNA was isolated directly from viral plaques and successfully analysed by PCR. No prior amplification of the virus by passage in tissue culture was necessary. The major advantage of this method was a reduction in assay times from a few days to a few hours. Moreover, this method is very convenient for detecting baculoviruses in cell culture: cross-contamination within viral stocks, monitoring mixed viral infection and confirmation of viral genomic integrity.
McCarthy
Christina B
CB
IBBM, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 1900 La Plata, Argentina. mccarthycb@agr.gc.ca <mccarthycb@agr.gc.ca>
Romanowski
Victor
V
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004279:DNA, Viral / Q000235:genetics* / Q000302:isolation & purification*; D004926:Escherichia coli; D007915:Lepidoptera; D011995:Recombination, Genetic; D015203:Reproducibility of Results
D004279:DNA, Viral