A simplified method of constructing infectious clones of begomovirus employing limited restriction enzyme digestion of products of rolling circle amplification.
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.
Wu
Chia-Ying
CY
Graduate Institute of Biotechnology, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 402, Taiwan.
Lai
Yi-Chin
YC
Lin
Na-Sheng
NS
Hsu
Yau-Heiu
YH
Tsai
Hsin-Tzu
HT
Liao
Jye-Yann
JY
Hu
Chung-Chi
CC
D053839:Begomovirus / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D016679:Genome, Viral; D010935:Plant Diseases / Q000821:virology*
D004262:DNA Restriction Enzymes