This protocol is a simple method for evaluating mutation frequency during African swine fever virus (ASFV) replication, although it could be used also for other DNA viruses (poxvirus, herpesvirus, mimivirus, etc) with minor modifications. In the original Carrascosa et al. (1982), the protocol was carried out with two cloned viruses, BA71Vc (a purified clone from BA71V wild type strain) and vΔpolX (lacking the reparative polymerase, pol X, gene), and two different cell types that can be infected by ASFV, Vero cells and swine macrophages. To facilitate the sequence comparison, a genome fragment containing the B646L gene was amplified by PCR and blunt-end cloned. This gene codes for the major capsid protein (p72) and multiple sequences can be found in the database, so the mutations found could be compared with natural gene variations. The cloned fragment can be either sequenced directly from bacteria colonies or from miniprep purified DNA.
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