Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis

Cleary, M. A., Kilian, K., Wang, Y., Bradshaw, J., Cavet, G., Ge, W., Kulkarni, A., Paddison, P. J., Chang, K., Sheth, N., Leproust, E., Coffey, E. M., Burchard, J., McCombie, W. R., Linsley, P., Hannon, G. J. (2004) Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis. Nat Methods, 1 (3). pp. 241-8.

URL: http://www.ncbi.nlm.nih.gov/pubmed/15782200
DOI: 10.1038/nmeth724

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.

Item Type: Paper
Additional Information:
Uncontrolled Keywords: Animals Cloning Molecular methods Computer Peripherals Humans In Situ Hybridization methods Mice Oligonucleotide Array Sequence Analysis methods Oligonucleotide Probes chemistry genetics Polymerase Chain Reaction methods RNA genetics
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing
bioinformatics > genomics and proteomics > design > nucleic acid design
CSHL Authors:
Communities: CSHL labs > Hannon lab
CSHL labs > McCombie lab
Watson School > Publications
Depositing User: CSHL Librarian
Date Deposited: 09 Feb 2012 16:24
Last Modified: 19 Sep 2014 14:56
Related URLs:
URI: http://repository.cshl.edu/id/eprint/22353

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