Application of Circular Polymerase Extension Cloning to Generate Infectious Clones of a Plant Virus

Abstract

Infectious clones of viruses allow elucidation of viral gene function in plants. This study presents an adaptation and expansion of the circular polymerase extension reaction that enables both cell-based and cell-free one-step assembly of a plant viral cDNA. To demonstrate its infectious nature, the generated clone was introduced into plants by Agrobacterium infiltration. This technique eliminates the cumbersome strategy of restriction enzyme digestion and ligation and instead relies on the sequence specificity of overlapping PCR fragments to assemble a complete functional infectious clone of a virus. This technique is rapid and potentially applicable for cloning virus genes which may be difficult to clone using conventional approaches due to toxicity problems that may be encountered when cloning in E. coli .