OligoMix® is a versatile, innovative, custom product for genomics discoveries. We synthesize thousands of oligonucleotide sequences in massive parallel on a microarray chip and then cleave the oligos, releasing them into solution in a single microtube. Synthesis occurs via standard DMT chemistry assuring efficient stepwise yield and a high quality final product. The product is delivered as a pool in a single microtube – ready for use in your experiment.
- Economical – At less than 0.8¢ per base, OligoMix is about 20 times more cost and time efficient than conventional oligos. Delivered in a single microtube, it enables inexpensive genome-scale experiments.
- Customizable – Customers can specify each oligonucleotide sequence (lengths up to 150-mers). We can synthesize oligonucleotides in OligoMix containing labels, such as terminus phosphate, amino and thiol with linkers, biotin, FAM or other dyes.
- Reliable – Innovative microfluidic array platform ensures high quality synthesis. Multiple QC steps are implemented at various stages of OligoMix manufacturing. OligoMix is subjected to both hybridization and qRT-PCR assays to assess final quality.
- Simple & Fast – Download our excel spreadsheet order form, paste in your sequences and email back to us. Product can be delivered in 1-2 weeks.
Microfluidic Array Platform—in situ Synthesis
OligoMix® achieves high synthesis purity because it is produced via an advanced microarray synthesis technology (µParaflo®) that integrates a photo-generated acid (PGA) chemistry, digital photolithography (DLP), and advanced microfluidics to enable high throughput parallel synthesis of custom DNA microarrays. The PGA chemistry enables the use of standard oligo building blocks, and eliminates the need for any specially modified nucleotides which may exhibit lower coupling efficiency. DLP technology enables programmable synthesis of custom sequences and the µParaflo® microfluidic device contains the synthesis reactions each within a picoliter-scale reaction chamber, producing more uniform synthesis than reactions performed on the open surface of a slide.
- Conventional Chemicals
- Established Synthesis Processes
- Efficient Stepwise Yield
- Quality Final Product
Synthesis Technology References
- Gao X, Yu PY, LeProust E, Sonigo L, Pellois JP, Zhang H. (1998) Oligonucleotide synthesis using solution photogenerated acids. Journal of the American Chemical Society 120, 12698-12699.
- Srivannavit O, Gulari M, Gulari E, LeProust E, Pellois JP, Gao X, Zhou X. (2004) Design and fabrication of microwell array chips for a solution-based, photogenerated acid-catalyzed parallel oligonucleotide DNA synthesis. Sensors and Actuators A. 116, 150-160.
- Zhou X, Cai S, Hong A, Yu P, Sheng N, Srivannavit O, Yong Q, Muranjan S, Rouillard JM, Xia Y, Zhang X, Xiang Q, Ganesh R, Zhu Q, Makejko A, Gulari E, Gao X. (2004) Microfluidic picoarray synthesis of oligodeoxynucleotides and simultaneously assembling of multiple DNA sequences. Nucleic Acids Research 32, 5409-5417.
- Tian J, Gong H, Sheng N, Zhou X, Gulari E, Gao X, Church G. (2004) Accurate multiplex gene synthesis from programmable DNA chips. Nature 432, 1050-1054 .
Library Construction Applications
Cloning complex libraries of reporter constructs with predefined nucleic acid sequences can be expensive and time-consuming. The use of synthetic oligonucleotides for sequence templates is a common practice when natural sources of the nucleic acid are not available, or a non-natural designed structure is desired. Traditionally, the oligos are synthesized individually, each on a separate solid support (column), and then individually engineered into the larger library. Because the synthesis and cloning steps are expensive and individual manipulation is labor intensive, this usually results in libraries with limited numbers of sequences (limited complexity).
OligoMix® enables us to synthesize thousands of designed sequences at once on a single microarray chip. By synthesizing the sequences in massive-parallel on a microfluidic chip, the overall cost and time required for synthesis is dramatically decreased and the cumbersome procedure of multiple transformation reactions for all unique constructs is avoided.
Production of cloned libraries with microarray-based oligo synthesis provides a rapid, high-throughput, cost-effective approach for the generation of complex libraries of designed oligo sequences. The flexibility to create completed designed custom sequences means this approach can address an array of biological questions, such as short hairpin RNA (shRNA) or CRISPR/Cas9 libraries for high-throughput loss-of-function genetic screens and antibody or other protein-coding DNA libraries for diversity studies and directed evolution strategies.
CRISPR/Cas9 Guide RNA Library Preparation
In CRISPR-Cas9 mutation screens, guide RNAs targeting tens of thousands of sites within genes are cloned into viral vectors and delivered as a pool into target cells along with Cas9. By identifying guide RNAs that are enriched or depleted in cells which exhibit a desired phenotype, researchers can systematically identify genes that are required for that particular phenotype.
In a typical guide RNA library prep strategy, oligos are individually synthesized on a microarray chip. These are then PCR amplified to incorporate vector compatible restriction sites, cloned into plasmid vectors (as above), and then transduced into viral libraries. Library pools of guides are then used for screening purposes. Following isolation of genomic DNA from positively selected cells, amplification by PCR across the guide region is performed and the guide identified by sequencing.1,2
Multiplexed Editing Regulatory Assay (MERA)3
In the MERA assay, a genomically integrated dummy guide RNA is replaced with a pooled library of guide RNAs through CRISPR-Cas9–based homologous recombination such that each cell receives a single guide RNA. Guide RNAs are tiled across the cis-regulatory regions of a GFP-tagged gene locus, and cells are flow cytometrically sorted according to their GFP expression levels. Deep sequencing on each population is used to identify guide RNAs preferentially associated with partial or complete loss of gene expression.
By scaling the guide RNA synthesis processes to massively parallel arrays, screening assays at unprecedented throughput are made possible. LC Sciences’ OligoMix® offers a unique solution for researchers looking to generate large guide RNA libraries, as users are able create fully designed libraries of thousands of specific, single-stranded oligonucleotide sequences for recognizing particular genomic regions.
Protein Coding / Antibody Library Preparation4,5
Synthetic antibody libraries have proven to be effective tools for drug discovery and development through the generation of functional, high-affinity antibodies against a wide variety of antigens. The performance of a synthetic antibody library depends in large part on the diversity of the library, which must be designed based on thorough understanding of the antibody structure and function. Insights from structural and functional analyses of functional antibodies are used to design synthetic oligonucleotides that introduce chemically and spatially defined diversity into the CDR loops. The synthetic CDR repertoires are incorporated into phage-display vectors to produce phage-displayed antibody repertoires.
|Custom synthesis of tens of thousands of specific (non-degenerate) oligo sequences enables the production of protein coding libraries with fully designed library diversity.|
|Specific sequences with any codon choices, deletions, insertions, and length variations are easily made.|
|The limitations of random codon generation are eliminated without expensive cost.|
While synthetic antibody libraries have many advantages such as optimized framework sequences and a broader sequence landscape than natural antibodies, their sequence diversities typically are generated by random combinatorial synthetic processes which cause the incorporation of many undesired CDR sequences. Researchers at Ewha Womans University have developed a method for construction of a synthetic scFv library using oligonucleotide mixtures that contain predefined, non-combinatorially synthesized CDR sequences6. Each CDR is first inserted to a master scFv framework sequence and the resulting single-CDR libraries are subjected to a round of proofread panning. The proofread CDR sequences are assembled to produce the final scFv library with six diversified CDRs.
Small Interfering RNA (siRNA) Library Preparation
RNAi for gene knock-down is a tool that has great promise. But beyond screening for basic identification of gene function, RNAi has the potential for therapeutic application via siRNA mediated gene silencing.
When considering siRNAs therapeutic potential, important issues that need to be addressed are siRNA specificity and analysis of any affect on off-target genes causing potential side-effects.
One approach is the use of an siRNA target library. A team led by researchers at the Karolinska Institute has developed a straightforward, efficient and cost effective method for generating an siRNA target library, by combining an siRNA target validation vector with OligoMix®7.
|Product Description||mix of DNA oligonucleotide sequences|
|Number of Oligos||thousands of sequences or more per tube|
|Oligo Form||single stranded (ss); desalted and ready for reaction|
|Length||up to 150 mers (inquire for longer oligos)|
|5’ or 3’ Terminus Modifications||phosphate, fluorescent dyes, biotin, linkers, and others|
|Internal Modifications||modified DNA or RNA bases|
|Yield||*tens of attomoles per sequence and a total of sub-fmols per OligoMix® tube|
- Malina A, Katigbak A, Cencic R, Maïga RI, Robert F, Miura H, Pelletier J. (2014) Adapting CRISPR/Cas9 for functional genomics screens. Methods Enzymol 546:193-213.
- Diao Y, Li B, Meng Z, Jung I, Lee AY, Dixon J, Maliskova L, Guan KL, Shen Y, Ren B. (2016) A new class of temporarily phenotypic enhancers identified by CRISPR/Cas9-mediated genetic screening. Genome Res 26(3):397-405.
- Rajagopal N, Srinivasan S, Kooshesh K, Guo Y, Edwards MD, Banerjee B, Syed T, Emons BJ, Gifford DK, Sherwood RI (2016) High throughput mapping of regulatory DNA. Nat Biotechnol 34(2):167-74.
- Xu M, Hu S, Ding B, Fei C, Wan W, Hu D, Du R, Zhou X, Hong J, Liu H. (2015) Design and construction of small perturbation mutagenesis libraries for antibody affinity maturation using massive microchip-synthesized oligonucleotides. Journal of Biotechnology 194(1), 27-36.
- Hu D, Hu S, Wan W, Xu M, Du R, Zhao W, Gao X, Liu J, Liu H, Hong J. (2015) Effective Optimization of Antibody Affinity by Phage Display Integrated with High-Throughput DNA Synthesis and Sequencing Technologies. PLoS ONE 10(6), e0129125.
- Bai X, Kim J, Kang S, Kim W, Shim H. (2015) A Novel Human scFv Library with Non-Combinatorial Synthetic CDR Diversity. PLoS ONE 10(10):e0141045.
- Dahlgren C, Zhang HY, Du Q, Grahn M, Norstedt G, Wahlestedt C, Liang Z. (2008) Analysis of siRNA specificity on targets with double-nucleotide mismatches. Nucleic Acids Res 36(9), e53.