A simple negative selection method to identify adenovirus recombinants using colony PCR |
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| Institution: | 1. Department of General Surgery, Southwest Hospital, Third Military Medical University, Chongqing 400038, China;2. Department of Medical Genetics, College of Basic Medicine, Third Military Medical University, Chongqing 400038, China |
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| Abstract: | BackgroundThe AdEasy system is a fast-track system for generating recombinant adenoviruses using the efficient homologous recombination machinery between shuttle and adenovirus backbone plasmids in Escherichia coli BJ5183 cells. The key step is homologous recombination in BJ5183 cells, which is driven by RecA activity. However, culture time is stringently limited to reduce the damage to recombinant plasmids by RecA activity. Therefore, rapid identification of recombinant adenoviruses within the limited time-period is critical.ResultsWe developed a simple negative selection method to identify recombinant adenoviruses using colony PCR, which improves the efficiency of adenovirus recombination screening and packaging.ConclusionsThe negative selection method to identify AdEasy adenovirus recombinants by colony PCR can identify the recombined colony within a short time-period, and maximally avoid damage to the recombinant plasmid by limiting recombination time, resulting in improved adenovirus packaging. |
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