Cloning biologically active geminivirus DNA using PCR and overlapping primers.

We report a new strategy for cloning DNA of the plant-infecting geminiviruses that is based on PCR amplification with primers overlapping the sequence of a restriction site in the viral genome. This method reduces the time and work necessary to obtain biologically active geminivirus DNAs and could be used for other DNA viruses having circular genomes, such as the animalinfecting circoviruses. Geminiviruses possess circular singlestranded DNA genomes that are monopartite [one DNA of ca. 2.6 kilobases (kb)] or bipartite (two DNAs designated DNA A and DNA B, each ca. 2.6 kb) and are encapsidated within twinned icosahedral particles (1). Replication of the viral genome occurs within plant cell nuclei via circular double-stranded replicative form (RF) DNA. RF DNA has been used to obtain infectious clones of several geminiviruses (2, 3, 4). Typically, RF DNA is isolated from infected plant tissue, a unique restriction site is identified within the DNA, and the digested full-length viral genome or genome component is cloned into a standard bacterial cloning vector. However, in our efforts to obtain full-length clones of the DNA A component of a bipartite tomato-infecting geminivirus from Costa Rica (given the provisional name TGVCR), we were unable to identify a suitable unique restriction site in the DNA A RF for cloning. Therefore, an alternative PCRbased strategy was developed.