Abstract
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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Acknowledgements
G.J.H. is supported by an Innovator Award from the US Army Breast Cancer Research Program. This work was also supported by a grant from the US National Institutes of Health (G.J.H.). We thank H. Dai for suggestions regarding microarray analysis of the library population, T. Fare for helpful comments on the manuscript, and the Rosetta Gene Expression Laboratory for microarray RNA processing and hybridizations.
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M.A.C., K.K., Y.W., J.B., G.C., W.G., A.K., E.M.C., J.B. and P.L. are employed by a for-profit company, Rosetta Inpharmatics, L.L.C., a wholly owned subsidiary of Merck, Inc. E.L. is an employee of Agilent Technologies. Partial funding for the work described was provided by Merck.
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Supplementary Fig. 1
Sequence traces of 390 oligonucleotides with a highly conserved core region of about ten consecutive bases (GGGTTGGCTC) that includes the shRNA loop structure. (PDF 163 kb)
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Cleary, M., Kilian, K., Wang, Y. et al. Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis. Nat Methods 1, 241–248 (2004). https://doi.org/10.1038/nmeth724
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DOI: https://doi.org/10.1038/nmeth724
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