A simplified method of constructing infectious clones of begomovirus employing limited restriction enzyme digestion of products of rolling circle amplification

• Published in: Journal of virological methods Vol. 147; no. 2; pp. 355 - 359
• Main Authors: Wu, Chia-Ying, Lai, Yi-Chin, Lin, Na-Sheng, Hsu, Yau-Heiu, Tsai, Hsin-Tzu, Liao, Jye-Yann, Hu, Chung-Chi
• Format: Journal Article
• Language: English
• Published: London Elsevier B.V 01.02.2008; Amsterdam Elsevier; New York, NY
• Subjects: Ageratum yellow vein virus; Begomovirus; Begomovirus - genetics; Biological and medical sciences; Cloning, Molecular - methods; DNA Restriction Enzymes - metabolism; Fundamental and applied biological sciences. Psychology; Genome, Viral; Infectious clone; Limited restriction enzyme digestion; Microbiology; Plant Diseases - virology; Rolling circle amplification; Squash leaf curl virus; Techniques used in virology; Tomato leaf curl virus; Virology; Infectious clone; Limited restriction enzyme digestion; Rolling circle amplification; Begomovirus; Plant pathogen; Microbiology; Enzyme; Method; Virology; Virus; Amplification; Infection; Geminiviridae
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2007.10.002

Most infectious clones of geminiviruses consist of (partial) tandem repeats of viral genomes in the vectors, which usually involve tedious, multi-step assemblies of genomic fragments in the construction process. A simplified procedure was devised to circumvent these problems, which employs limited restriction digestion of multimeric viral genomes produced by rolling circle amplification (RCA), followed by direct cloning into appropriate vectors. The efficiency of the procedure, and infectivity of the dimeric constructs it produced, were demonstrated using three different geminiviruses, namely ageratum yellow vein virus, tomato leaf curl virus, and squash leaf curl virus.