The Revolution of Oligo Pools in High-Throughput Genomics

Oligo pools are emerging as a revolutionary tool for high-throughput genomics experiments. As defined by Dynegene, oligo pools are complex mixtures of synthetic oligonucleotides (short DNA fragments) that enable rapid and cost-effective construction of diverse DNA libraries for screening and selection. Compared to traditional gene synthesis methods, oligo pools provide researchers access to thousands-millions of unique DNA sequences at a fraction of the cost. This article explores the technical details behind oligo pool construction and highlights cutting-edge applications of this transformative technology.

Oligo Pool Construction

The creation of high-quality oligo pools relies on scalable, high-fidelity DNA synthesis. As a leader in ultra-high-throughput DNA synthesis, Dynegene has developed proprietary DYHOW technology to construct DNA oligos up to 230 nucleotides long with accuracy exceeding 99.7%. These oligos are synthesized in situ as programmable printheads deposit nucleotides onto custom microarrays, achieving unparalleled scale and precision.

To construct an oligo pool, Dynegene leverages computational algorithms to design libraries of thousands to millions of unique oligo sequences. These oligos are then massively synthesized in parallel on arrays containing millions of distinct features. Following synthesis, oligos are cleaved from the array and PCR amplified to increase quantities. The resulting complex pools contain an adjustable range of oligo lengths and concentrations to suit different applications.

Quality Control of Oligo Pools

Verifying the quality of oligo pools is critical prior to use in downstream experiments. Dynegene implements several quality control steps throughout the construction pipeline. During synthesis, each nucleotide addition is monitored in real-time using computer vision algorithms. This enables early detection of synthesis errors.

Following amplification, next-generation sequencing provides base-resolution insights into oligo pool quality. Bioinformatics analysis confirms library diversity, measures oligo lengths/concentrations, and identifies any erroneous sequences. Customers can request detailed sequencing reports to validate performance prior to purchasing.

Overall, Dynegene's automated manufacturing processes facilitate rapid QC testing and ensure batch-to-batch consistency. This gives researchers confidence in using oligo pools across diverse projects.

Advancing Protein Engineering with Oligo Pools

One major application of oligo pools is constructing DNA libraries for protein engineering. Traditionally, this required laborious gene-by-gene synthesis of mutant libraries. In contrast, oligo pools enable one-step construction of libraries with billions of sequence variants. For example, Dynegene offers Custom Mutagenesis Pools up to 230bp long for saturating all possible amino acid mutations across a protein target.

Constructing these oligo pools is as simple as submitting a target protein sequence. Dynegene's proprietary Diversity Algorithm then designs oligos spanning the full gene while encoding every possible single, double, or triple mutation combination. Following array-based synthesis and amplification, the oligo pools are sequence-verified and ready for library construction through Gibson assembly or other molecular cloning techniques.

Researchers can then screen these vast protein mutation libraries to discover variants with enhanced stability, altered substrate specificity, or improved catalytic properties. Oligo pools accelerate protein engineering campaigns from months to weeks, enabling rapid iteration.

Empowering Genome Editing with Oligo Pools

In addition to protein engineering, oligo pools are invaluable for constructing diverse donor libraries for CRISPR genome editing experiments. By synthesizing pools of repair templates or single guide RNAs (sgRNAs), researchers can massively test editing conditions in parallel. For example, oligo pools containing up to 100,000 unique sgRNAs have been used for pooled CRISPR knockout screening.

Dynegene offers Custom CRISPR Libraries with variable sgRNA designs tailored to each project. After selecting genomic target regions, Dynegene's bioinformatics pipeline identifies candidate sgRNAs, avoiding off-targets. These sgRNAs are oligo pool synthesized and amplified complete with cloning adapters, enabling direct Gibson Assembly into lentiviral constructs for pooled screening.

The simplicity of producing ready-to-use sgRNA pools eliminates the burden of individual sgRNA cloning. Genome editing experiments also benefit from the diversity and scale uniquely achievable with oligo pools. This expands the breadth of testable conditions to accelerate discovery.

Oligo Pools for Drug Discovery

Oligo pools further enable high-throughput drug discovery by allowing construction of vast one-bead-one-compound (OBOC) libraries. Here, oligos are synthesized containing a chemical reaction sequence, linker, and encoding barcode. Oligos are then PCR amplified and transcribed to RNA which directs the stepwise chemical coupling reactions onto beads presenting a starting scaffold.

Millions of beads, each displaying a unique small molecule variant, can be rapidly synthesized in this manner. Pooled screening then identifies beads displaying compounds with desired bioactivity by sequencing the barcode. Hit compound structures can be decoded from the originating oligonucleotide sequences.

Dynegene provides Custom OBOC Libraries synthesized by this approach to empower drug discovery. By supplying chemically diverse DNA-encoded compound libraries containing upwards of 100 million members, Dynegene enables high-throughput lead generation and target binding studies.

The Future of Oligo Pools

Oligo pools are an increasingly vital tool for constructing the vast DNA libraries underlying rapid, massively parallel experiments in synthetic biology. As described above, applications span protein engineering, genome editing, drug discovery, and beyond. However, oligo pools are not limited to these areas.

The customizability and scalability of high-fidelity oligo synthesis opens new doors for designing and testing diverse nucleic acid libraries. For example, future oligo pool applications may include optimizing RNA therapeutics, evolving novel genetic circuits, or prototyping complex DNA nanostructures.

Thanks to advances in ultra-high-throughput array-based DNA synthesis from innovators like Dynegene, researchers now have unparalleled access to customized oligo pools. This democratization promises to accelerate discovery across genomics, synthetic biology, and molecular engineering.

Conclusion

The advent of oligo pools represents a revolution in constructing libraries for high-throughput biology. Compared to traditional DNA synthesis methods, oligo pools enable massively parallel testing of orders of magnitude more sequence variants - whether for protein engineering, genome editing, drug discovery, or beyond.

Backed by leaders like Dynegene with expertise in ultra-high-fidelity, ultra-high-throughput oligo synthesis, researchers across disciplines now have access to this transformative technology. The customizability and scalability of oligo pools promises to supercharge experimental throughput and discovery across genomics and synthetic biology. By expanding testable possibilities, oligo pools exemplify the coming wave of high-throughput biological experimentation.