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


RESULTS
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.


CONCLUSIONS
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.
Biotechnology Monsanto Company, St. Louis, Missouri, United States of America. Brian.hauge@monsanto.com
Brian
B
Hauge
Christopher
C
Oggero
Nicole
N
Nguyen
Changlin
C
Fu
Fenggao
F
Dong
D017360:Arabidopsis / Q000235:genetics*; D003001:Cloning, Molecular; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D020868:Gene Silencing; D005821:Genetic Techniques; D005822:Genetic Vectors; D016376:Oligonucleotides, Antisense / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000378:metabolism; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics*; D014498:Uracil / Q000737:chemistry
D016376:Oligonucleotides, Antisense; D012330:RNA, Double-Stranded; D014498:Uracil; D012313:RNA