Single tube, high throughput cloning of inverted repeat constructs for double-stranded RNA expression

• Published in: PloS one Vol. 4; no. 9; p. e7205
• Main Authors: Hauge, Brian, Oggero, Christopher, Nguyen, Nicole, Fu, Changlin, Dong, Fenggao
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 28.09.2009; Public Library of Science (PLoS)
• Subjects: Annealing; Antisense RNA; Arabidopsis - genetics; Assembly; Biotechnology; Biotechnology/Plant Biotechnology; Cloning; Cloning, Molecular; Deoxyribonucleic acid; DNA; DNA polymerase; Double-stranded RNA; Drug discovery; Electrophoresis, Agar Gel - methods; Enzymes; Escherichia coli - genetics; Fragments; Gene expression; Gene Silencing; Genetic research; Genetic Techniques; Genetic Vectors; Genetics and Genomics; Genetics and Genomics/Chromosome Biology; Genetics and Genomics/Epigenetics; Genetics and Genomics/Functional Genomics; Genetics and Genomics/Gene Discovery; Genetics and Genomics/Gene Expression; Genetics and Genomics/Gene Function; Genetics and Genomics/Genomics; Genetics and Genomics/Plant Genetics and Gene Expression; Genetics and Genomics/Plant Genomes and Evolution; Genomes; Genomics; High-throughput screening; In vivo methods and tests; Inverted repeat; Ligases; Molecular biology; Mutagenesis; Oligonucleotides, Antisense - genetics; Plants (botany); Plants, Genetically Modified - genetics; Polymerase chain reaction; Polymerase Chain Reaction - methods; Ribonucleic acid; RNA; RNA - metabolism; RNA Interference; RNA, Double-Stranded - genetics; RNA-mediated interference; Uracil - chemistry; United States > US; Missouri
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0007205

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. 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. 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.