Combinatorial PCR Method for Efficient, Selective Oligo Retrieval from Complex Oligo Pools

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. The selective retrieval of specific oligos from these complex pools traditionally uses polymerase chain reaction (PCR). Designing a large number of primers to use in PCR presents a serious challenge, particularly for DNA data storage, where the size of an oligo pool is orders of magnitude larger than other applications. Although a nested primer address system was recently developed to increase the number of accessible files for DNA storage, it requires more complicated lab protocols and more expensive reagents to achieve high specificity, as well as more DNA address space. Here, we present a new combinatorial PCR method that has none of those drawbacks and outperforms in retrieval specificity. In experiments, we accessed three files that each comprised 1% of a DNA prototype database that contained 81 different files and enriched them to over 99.9% using our combinatorial primer method. Our method provides a viable path for scaling up DNA data storage systems and has broader utility whenever one must access a specific target oligo and can design their own primer regions.

Emerging Approaches to DNA Data Storage: Challenges and Prospects

“…Nonetheless, primer-based addressing systems face several constraints. First, the incorporation of random-access priming regions into each oligo decreases the available space for data-encoding bases, thereby also decreasing the storage density (currently by about 15% per address region). , Second, PCR-based random access irreversibly removes oligos from the pool, which necessitates potentially lossy reamplification of the entire pool after repeated data retrieval. , 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. , Indeed, data retrieval from a hierarchical addressing system of 5.5 TB required additional physical separation of pools via a biotin-based bead extraction between file accesses to fully remove the background carried over from PCR . Lastly, PCR-based addressing is incompatible with common storage approaches, thus necessitating the removal and re-embedding of the encoding DNA into the storage matrix for each random-access operation.…”

“… 75 , 76 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 Indeed, data retrieval from a hierarchical addressing system of 5.5 TB required additional physical separation of pools via a biotin-based bead extraction between file accesses to fully remove the background carried over from PCR. 71 Lastly, PCR-based addressing is incompatible with common storage approaches, thus necessitating the removal and re-embedding of the encoding DNA into the storage matrix for each random-access operation.…”

The DNA Data Storage Model

“…There are a variety of methods to implement such operations, but two popular methods are PCR 3,13,14 and DNA pull-out with magnetic nanoparticles 15 (Figure 9).…”

OligoArchive-DSM: Columnar Design for Error-Tolerant Database Archival using Synthetic DNA

“…However, it is often necessary to retrieve only a small amount of data. Prior work has achieved this by assuming an object-based get/put interface to DNA storage and relying on the use of PCR for achieving random access of individual objects [9], [34]. The central idea is to associate a distinct pair of short DNA sequence, also called primers, to all oligos belonging to each distinct object.…”

“…However, there has been limited work on systematically quantifying coverage bias introduced by PCR-based random access [34]. In practical archival scenarios, objects stored tend to have widely varying sizes.…”