Wu Chia-Ying, Lai Yi-Chin, Lin Na-Sheng, Hsu Yau-Heiu, Tsai Hsin-Tzu, Liao Jye-Yann, Hu Chung-Chi
Graduate Institute of Biotechnology, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 402, Taiwan.
J Virol Methods. 2008 Feb;147(2):355-9. doi: 10.1016/j.jviromet.2007.10.002. Epub 2007 Nov 26.
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.
大多数双生病毒的感染性克隆由载体中病毒基因组的（部分）串联重复组成，这在构建过程中通常涉及基因组片段繁琐的多步组装。设计了一种简化程序来规避这些问题，该程序采用对滚环扩增（RCA）产生的多聚体病毒基因组进行有限的限制性消化，然后直接克隆到合适的载体中。使用三种不同的双生病毒，即藿香蓟黄脉病毒、番茄卷叶病毒和南瓜卷叶病毒，证明了该程序的效率及其产生的二聚体构建体的感染性。
