Rapid method for the isolation of full length adenoviral genomes by bacterial intermolecular homologous recombination.
Recombinant adenoviruses are used widely in gene therapy research. Much work has been carried out to remove specific components of the wild type adenovirus (e.g. E1 gene) in order to make them safer for human use. In addition to such efforts, it is vitally important to ensure that the production of recombinant adenoviruses meet safety guidelines not only with regard to the absence of replication competent adenoviruses but for other variant species that may be present in a viral preparation. In this report, a time and cost efficient method is described for the isolation of full length adenovirus genomes without resorting to plaque purification. The procedure uses a bacterial homologous recombination system and results in the conversion of the double-stranded linear adenovirus genome into a circularized plasmid form that can be easily analyzed by restriction digestion, PCR, DNA sequencing or used in transient transfection studies. Also, the adenovirus plasmids that are generated may also be rescued back into virus form if needed. The entire procedure takes 4 days or less instead of weeks that plaque purification or dilution cloning requires. Furthermore, the method does not require the use of tissue culture materials or facilities. More importantly, this procedure allows for a more extensive and thorough examination of any viral preparation, since it allows for the detection of variants incapable of propagation without the assistance of co-infecting intact adenoviral genomes. Under standard conditions of plaque purification, these variant genomes are not detected. It is predicted that far more variant genomes will be observed using this rapid method than would otherwise be detected by standard plaque purification methods.
Youil
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R
Merck and Co., Department of Virus and Cell Biology, West Point, PA 19486, USA. rima_youil@merck.com
Toner
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TJ
Su
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Kaslow
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D000256:Adenoviridae / Q000235:genetics*; D002999:Clone Cells; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D016679:Genome, Viral; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D014162:Transfection
D004279:DNA, Viral