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Methods Enzymol. 2005;392:405-19.

Simple, robust strategies for generating DNA-directed RNA interference constructs.

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Benitec Ltd., University of Queensland, St. Lucia QLD, Australia.


We describe two complementary strategies for preparing DNA-directed RNA interference (ddRNAi) constructs designed to express hpRNA. The first, oligonucleotide assembly (OA), uses a very simple annealing protocol to combine up to 20 short nucleotides. These are then cloned into appropriately designed restriction sites in expression vectors. OA can be used to prepare simple hairpin (hp)-expressing constructs, but we prefer to use the approach to generate longer constructs. The second strategy, long-range cloning (LRC), uses a novel adaptation of long-range PCR protocols. For LRC, entire vectors are amplified with primers that serve to introduce short sequences into plasmids at defined anchor sites during PCR. The LCR strategy has proven highly reliable in our hands for generating simple ddRNAi constructs. Moreover, LCR is likely to prove useful in many situations in which conventional cloning strategies might prove problematic. In combination, OA and LRC can greatly simplify the design and generation of many expression constructs, including constructs for ddRNAi.

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