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Nucleic Acids Res. 2010 May;38(8):2522-40. doi: 10.1093/nar/gkq163. Epub 2010 Mar 22.

Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process.

Author information

1
Agilent Technologies Inc., LSSU - Genomics, 5301 Stevens Creek Blvd, Santa Clara, CA 95051, USA. emily_leproust@agilent.com

Abstract

We have achieved the ability to synthesize thousands of unique, long oligonucleotides (150mers) in fmol amounts using parallel synthesis of DNA on microarrays. The sequence accuracy of the oligonucleotides in such large-scale syntheses has been limited by the yields and side reactions of the DNA synthesis process used. While there has been significant demand for libraries of long oligos (150mer and more), the yields in conventional DNA synthesis and the associated side reactions have previously limited the availability of oligonucleotide pools to lengths <100 nt. Using novel array based depurination assays, we show that the depurination side reaction is the limiting factor for the synthesis of libraries of long oligonucleotides on Agilent Technologies' SurePrint DNA microarray platform. We also demonstrate how depurination can be controlled and reduced by a novel detritylation process to enable the synthesis of high quality, long (150mer) oligonucleotide libraries and we report the characterization of synthesis efficiency for such libraries. Oligonucleotide libraries prepared with this method have changed the economics and availability of several existing applications (e.g. targeted resequencing, preparation of shRNA libraries, site-directed mutagenesis), and have the potential to enable even more novel applications (e.g. high-complexity synthetic biology).

PMID:
20308161
PMCID:
PMC2860131
DOI:
10.1093/nar/gkq163
[Indexed for MEDLINE]
Free PMC Article

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