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

Michele A. Cleary, Kristopher Kilian, Yanqun Wang, Jeff Bradshaw, Guy Cavet, Wei Ge, Amit Kulkarni, Patrick J. Paddison, Kenneth Chang, Nihar Sheth, Eric Leproust, Ernest M. Coffey, Julja Burchard, W. Richard McCombie, Peter Linsley, Gregory J. Hannon

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Pages (from-to)241-248
Number of pages8
JournalNature Methods
Issue number3
StatePublished - Dec 2004
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology


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