Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process
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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).
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Conventional phosphoramidite DNA synthesis relies on an acidic reagent such as trichloroacetic acid to remove the DMT protecting group at the 5′ end to activate the nucleotide monomer.
“Conventional DNA synthesis based on the phosphoramidite chemistry relies on an acidic reagent (e.g., trichloroacetic acid) to remove the protecting group [e.g., dimethyltrityl (DMT)] at the 5′ end to activate the nucleotide monomer (34).”
From Electrochemical DNA synthesis and sequencing on a single electrode with scalability for integrated data storage (2021) · cites this paper as [34]
During synthesis of DNA, growing strands may be terminated before reaching desired length and thus induce a bias to the pool.
“During synthesis of DNA, growing strands may be terminated before having reached the desired length and thus induce a bias to the pool36,37.”
From DNA synthesis for true random number generation (2020) · cites this paper as [36]
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References 59 · Cited by 326
- Authors
- LeProust, E. M., Peck, B. J., Spirin, K., McCuen, H. B., Moore, B., Namsaraev, E., & Caruthers, M. H.
- Affiliations
- Agilent Technologies (United States) (US)
- Corresponding author
- LeProust, Emily M.
- ORCID
- Caruthers, Marvin H.
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 2010-03-20
- Volume
- 38
- Issue
- 8
- Pages
- 2522-2540
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- PMC
- PMC2860131
- OpenAlex
- W2109243615
- Cited by
- 326
- References
- 59
- Citations by year
- 2026: 17 · 2025: 22 · 2024: 32 · 2023: 17 · 2022: 16 · 2021: 17 · 2020: 23 · 2019: 26 · 2018: 24 · 2017: 13 · 2016: 18 · 2015: 25 · 2014: 16 · 2013: 20 · 2012: 23
- FWCI
- 7.68
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Nucleic Acids Research (publishedVersion, cc-by-nc)
- PubMed Central (publishedVersion, cc-by-nc)
- PubMed (publishedVersion)
- CU Scholar (University of Colorado Boulder) (submittedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced biosensing and bioanalysis techniques
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Keywords
- oligonucleotide libraries, depurination, detritylation, synthesis yield, targeted sequencing, shRNA library, site-directed mutagenesis, synthetic biology
- MeSH terms
- Indicators and Reagents, Nucleic Acid Hybridization, Oligonucleotides, Purines, Polymerase Chain Reaction, Oligonucleotide Array Sequence Analysis
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