Combinatorial PCR Method for Efficient, Selective Oligo Retrieval from Complex Oligo Pools
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Abstract
ABSTRACT With the rapidly decreasing cost of array-based oligo synthesis, large-scale oligo pools offer significant benefits for advanced applications, including gene synthesis, CRISPR-based gene editing, and DNA data storage. Selectively retrieving specific oligos from these complex pools traditionally uses Polymerase Chain Reaction (PCR), in which any selected oligos are exponentially amplified to quickly outnumber non-selected ones. In this case, the number of orthogonal PCR primers is limited due to interactions between them. This lack of specificity presents a serious challenge, particularly for DNA data storage, where the size of an oligo pool (i.e., a DNA database) is orders of magnitude larger than it is for other applications. Although a nested file address system was recently developed to increase the number of accessible files for DNA storage, it requires a more complicated lab protocol and more expensive reagents to achieve high specificity. Instead, we developed a new combinatorial PCR method that outperforms prior work without compromising the fidelity of retrieved material or complicating wet lab processes. Our method quadratically increases the number of accessible oligos while maintaining high specificity. In experiments, we accessed three arbitrarily chosen files from a DNA prototype database that contained 81 different files. Initially comprising only 1% of the original database, the selected files were enriched to over 99.9% using our combinatorial primer method. Our method thus provides a viable path for scaling up DNA data storage systems and has broader utility whenever scientists need access to a specific target oligo and can design their own primer regions.
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As pool sizes and number of sequences become larger, enrichment of a few copies against an ever-increasing background will at some point hit limitations of PCR regarding processing volumes, required amplification cycles, and nonspecific amplification due to primer-payload similarity.
“Moreover, as pool sizes and, thus, the number of sequences, become larger, the enrichment of a few copies against an ever-increasing background will at some point hit the limitations of PCR regarding processing volumes, required amplification cycles to obtain sufficient enrichment, and nonspecific amplification due to primer–payload similarity.77,70”
From Emerging Approaches to DNA Data Storage: Challenges and Prospects (2022) · cites this paper as [77]
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Emerging Approaches to DNA Data Storage: Challenges and Prospects
Doricchi, A., Platnich, C. M., Gimpel, A., Horn, F., Earle, M., Lanzavecchia, G., Cortajarena, A. L., Liz-Marzán, L. M., Liu, N., Heckel, R., et al. (2022) · ACS Nano · doi:10.1021/acsnano.2c06748 · Cited by 235 · see citing works
- Authors
- Winston, C., Organick, L., Ward, D., Ceze, L., Strauss, K., & Chen, Y.-J.
- Affiliations
- University of Washington (US)
- Paul G. Allen School of Computer Science and Engineering, University of Washington, Seattle, Washington 98195, United States
- Microsoft (United States) (US)
- Corresponding author
- Chen, Yuan-Jyue
- Venue
- ACS Synthetic Biology
- Publisher
- American Chemical Society (ACS)
- Published
- 2022-02-22
- Volume
- 11
- Issue
- 5
- Pages
- 1727-1734
- ISSN
- 2161-5063 (ISSN-L)
- OpenAlex
- W4213203727
- Cited by
- 29
- References
- 24
- Citations by year
- 2026: 5 · 2025: 7 · 2024: 9 · 2023: 6 · 2022: 2
- FWCI
- 1.96
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- ACS Synthetic Biology (publishedVersion, cc-by-nc-nd)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nd)
- PubMed (publishedVersion)
- Funders
- Defense Advanced Research Projects Agency
- Microsoft
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Biological Computing
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced biosensing and bioanalysis techniques
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Keywords
- DNA data storage, primer design, gene synthesis, CRISPR gene editing
- MeSH terms
- DNA, Polymerase Chain Reaction, Information Storage and Retrieval, DNA Primers, Databases, Nucleic Acid
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