IBBM   21076
INSTITUTO DE BIOTECNOLOGIA Y BIOLOGIA MOLECULAR
Unidad Ejecutora - UE
artículos
Título:
A simplified method for the extraction of baculoviral DNA for PCR analysis: a practical application
Autor/es:
MCCARTHY C.B. & V. ROMANOWSKI
Revista:
JOURNAL OF VIROLOGICAL METHODS
Referencias:
Año: 2008 vol. 148 p. 286 - 290
ISSN:
0166-0934
Resumen:
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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.