title: Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis.
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.
authors.affiliation: Rosetta Inpharmatics LLC, a wholly owned subsidiary of Merck & Co., Inc., 401 Terry Ave. North, Seattle, Washington 98109, USA.
authors.forename: Michele A
authors.initials: MA
authors.lastname: Cleary
authors.forename: Kristopher
authors.initials: K
authors.lastname: Kilian
authors.forename: Yanqun
authors.initials: Y
authors.lastname: Wang
authors.forename: Jeff
authors.initials: J
authors.lastname: Bradshaw
authors.forename: Guy
authors.initials: G
authors.lastname: Cavet
authors.forename: Wei
authors.initials: W
authors.lastname: Ge
authors.forename: Amit
authors.initials: A
authors.lastname: Kulkarni
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Paddison
authors.forename: Kenneth
authors.initials: K
authors.lastname: Chang
authors.forename: Nihar
authors.initials: N
authors.lastname: Sheth
authors.forename: Eric
authors.initials: E
authors.lastname: Leproust
authors.forename: Ernest M
authors.initials: EM
authors.lastname: Coffey
authors.forename: Julja
authors.initials: J
authors.lastname: Burchard
authors.forename: W Richard
authors.initials: WR
authors.lastname: McCombie
authors.forename: Peter
authors.initials: P
authors.lastname: Linsley
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Hannon
mesh_terms: D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D016287:Computer Peripherals; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000235:genetics
chemical_list: D015345:Oligonucleotide Probes; D012313:RNA