Nature Methods1, 241 - 248 (2004)
Published online: 18 November 2004; | doi:10.1038/nmeth724
Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis
Michele A Cleary1, Kristopher Kilian1, Yanqun Wang1, Jeff Bradshaw1, Guy Cavet1, Wei Ge1, Amit Kulkarni1, Patrick J Paddison2, Kenneth Chang2, Nihar Sheth2, Eric Leproust3, Ernest M Coffey1, Julja Burchard1, W Richard McCombie2, Peter Linsley1
& Gregory J Hannon2
1
Rosetta Inpharmatics LLC, a wholly owned subsidiary of Merck & Co., Inc., 401 Terry Ave. North, Seattle, Washington 98109, USA.
2
Cold Spring Harbor Laboratory, Watson School of Biological Sciences, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.
3
Agilent Technologies, 3500 Deer Creek Road, Palo Alto, California 94304, USA.
Generation of complex libraries of defined nucleic acid sequences can greatly aid the functional analysis of protein and gene function. Previously, such studies relied either on individually synthesized oligonucleotides or on cellular nucleic acids as the starting material. As each method has disadvantages, we have developed a rapid and cost-effective alternative for construction of small-fragment DNA libraries of defined sequences. This approach uses in situ microarray DNA synthesis for generation of complex oligonucleotide populations. These populations can be recovered and either used directly or immortalized by cloning. From a single microarray, a library containing thousands of unique sequences can be generated. As an example of the potential applications of this technology, we have tested the approach for the production of plasmids encoding short hairpin RNAs (shRNAs) targeting numerous human and mouse genes. We achieved high-fidelity clone retrieval with a uniform representation of intended library sequences.
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