PCR-based plasmid vector construction for generation of recombinant viruses.
A totally polymerase chain reaction (PCR)-based protocol for construction of plasmids for production of recombinant macropodid herpesvirus 1 (MaHV-1) is described. This protocol greatly simplifies traditional methods that use restriction enzyme-based cloning or a combination of restriction enzyme cloning and the PCR. PCR is used to amplify the vector backbone containing an origin of replication and selectable marker, and the inserts to be cloned (5' and 3' viral homologous recombination regions and the reporter gene green fluorescent protein (GFP)). The inserts are cloned in a sequential manner with the intermediate vectors then amplified to produce the next vector. At its most basic, this involves, after the initial PCR amplification of vector and inserts, two additional PCR amplifications and three ligation events. This protocol is however totally generic, and can be used not only for construction of plasmids for production of recombinant viruses, but also for any general cloning applications.