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Updated: Apr 23, 2026

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses
Published on: December 29, 2015
Simplified methods for the construction of RNA and DNA virus infectious clones
Tatsuya Nagata1, Alice Kazuko Inoue-Nagata
1Laboratório de Microscopia e Virologia, Departamento de Biologia Celular, Conselho de Ensino, Pesquisa, e Extensão, Universidade de Brasília, IB-Bloco K, Asa Norte, 70910-900, Brasilia, DF, Brazil, tatsuya@unb.br.
Abstract:
Infectious virus clones are one of the most powerful tools in plant pathology, molecular biology, and biotechnology. The construction of infectious clones of RNA and DNA viruses, however, usually requires laborious cloning and subcloning steps. In addition, instability of the RNA virus genome is frequently reported after its introduction into the vector and transference to Escherichia coli. These difficulties hamper the cloning procedures, making it tedious and cumbersome. This chapter describes two protocols for a simple construction of infectious viruses, an RNA virus, the tobamovirus Pepper mild mottle virus, and a DNA virus, a bipartite begomovirus. For this purpose, the strategy of overlap-extension PCR was used for the construction of infectious tobamovirus clone and of rolling circle amplification (RCA) for the construction of a dimeric form of the begomovirus clone.
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