single tube, high throughput cloning of inverted repeat constructs for double-stranded rna expression单管,高通量克隆反向重复结构的双链rna表达.pdfVIP

Single Tube, High Throughput Cloning of Inverted Repeat Constructs for Double-Stranded RNA Expression Brian Hauge*, Christopher Oggero, Nicole Nguyen, Changlin Fu, Fenggao Dong Biotechnology Monsanto Company, St. Louis, Missouri, United States of America Abstract Background: RNA interference (RNAi) has emerged as a powerful tool for the targeted knockout of genes for functional genomics, system biology studies and drug discovery applications. To meet the requirements for high throughput screening in plants we have developed a new method for the rapid assembly of inverted repeat-containing constructs for the in vivo production of dsRNAs. Methodology/Principal Findings: The procedure that we describe is based on tagging the sense and antisense fragments with unique single-stranded (ss) tails which are then assembled in a single tube Ligase Independent Cloning (LIC) reaction. Since the assembly reaction is based on the annealing of unique complementary single stranded tails which can only assemble in one orientation, greater than ninety percent of the resultant clones contain the desired insert. Conclusion/Significance: Our single-tube reaction provides a highly efficient method for the assembly of inverted repeat constructs for gene suppression applications. The single tube assembly is directional, highly efficient and readily adapted for high throughput applications. Citation: Hauge B, Oggero C, Nguyen N, Fu C, Dong F (2009) Single Tube, High Throughput Cloning of Inverted Repeat Constructs for Double-Stranded RNA Expression. PLoS ONE 4(9): e7205. doi:10.1371/journal.pone.0007205 Editor: Magdalena Bezanilla, University of Massachusetts Amherst, United States of America Received May 22, 2009; Accepted August 26, 2009; Published September 28, 2009 Copyright: 2009 Hauge et al. This is an open-access article