Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis.
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.
Cleary
Michele A
MA
Rosetta Inpharmatics LLC, a wholly owned subsidiary of Merck & Co., Inc., 401 Terry Ave. North, Seattle, Washington 98109, USA.
Kilian
Kristopher
K
Wang
Yanqun
Y
Bradshaw
Jeff
J
Cavet
Guy
G
Ge
Wei
W
Kulkarni
Amit
A
Paddison
Patrick J
PJ
Chang
Kenneth
K
Sheth
Nihar
N
Leproust
Eric
E
Coffey
Ernest M
EM
Burchard
Julja
J
McCombie
W Richard
WR
Linsley
Peter
P
Hannon
Gregory J
GJ
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
D015345:Oligonucleotide Probes; D012313:RNA