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

Rice, Robert R., Muirhead, Andrew N., Harrison, Bruce T., Kassianos, Andrew J., Sedlak, Petra L., Maugeri, Narelle J., Goss, Peter J., Davey, Jonathan R., James, David E. and Graham, Michael W. (2005) Simple, robust strategies for generating DNA-directed RNA interference constructs. Methods in Enzymology, 392 18: 405-419. doi:10.1016/S0076-6879(04)92024-1

Author Rice, Robert R.
Muirhead, Andrew N.
Harrison, Bruce T.
Kassianos, Andrew J.
Sedlak, Petra L.
Maugeri, Narelle J.
Goss, Peter J.
Davey, Jonathan R.
James, David E.
Graham, Michael W.
Title Simple, robust strategies for generating DNA-directed RNA interference constructs
Journal name Methods in Enzymology   Check publisher's open access policy
ISSN 0076-6879
ISBN 978-0-12-182797-7
Publication date 2005-01-01
Year available 2011
Sub-type Article (original research)
DOI 10.1016/S0076-6879(04)92024-1
Open Access Status Not yet assessed
Volume 392
Issue 18
Start page 405
End page 419
Total pages 15
Editor David R. Engelke
John J. Rossi
Place of publication New York, U.S.A.
Publisher Academic Press
Language eng
Subject 1101 Medical Biochemistry and Metabolomics
Abstract 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. Copyright © 2004 Elsevier Inc
Keyword RNA interference constructs
Long range cloning
Q-Index Code C1
Grant ID R01 GM080784
Institutional Status Non-UQ
Additional Notes Monographic series. Volume title: RNA Interference

Document type: Journal Article
Sub-type: Article (original research)
Collections: Excellence in Research Australia (ERA) - Collection
Queensland Brain Institute Publications
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Citation counts: TR Web of Science Citation Count  Cited 6 times in Thomson Reuters Web of Science Article | Citations
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Created: Mon, 11 Jan 2010, 22:58:33 EST by Natalie Holt on behalf of Queensland Brain Institute