Cartridge (OPC)-based Oligonucleotide Fragment Purification Service
The Importance of Oligonucleotide Purification Cartridge (OPC)-based Oligonucleotide Fragment Purification
Modern DNA-based methods require large-scale production of oligonucleotides. Automated systems operating in an open microtiter plate format can synthesize thousands of oligonucleotides in a highly parallel fashion. Arrays made using pre-synthesized and spotted oligodeoxynucleotides (ODNs) typically consist of thousands of oligodeoxynucleotides. Optimal sensitivity and specificity have been achieved with probes ranging in length from 50 to 70 bases. However, various by-products may contaminate oligodeoxynucleotide preparations. Foremost among these are truncated fragments, these by-products resulting from inefficient coupling steps that lead to product capping and depurination processes. These processes finally lead to strand scission upon ammonia treatment. Due to their sequential nature, traditional oligonucleotide separation methods are not well suited for the rapid purification of thousands of different products. The need is therefore addressed by efficient OPG-based purification methods that allow for the development of techniques for oligonucleotide synthesis and deprotection.
Key Technologies
At CD BioGlyco, our OPC-based purification leverages reverse-phase (RP) chromatography columns that exploit the specific affinity between resin and DMT groups to isolate full-length oligonucleotides. We selectively bind DMT-retaining fragments, wash away failure sequences, then cleave DMT groups with trifluoroacetic acid before eluting high-purity products. This optimized process ensures efficiency, scalability, and reliability for PCR primers, probes, and sequencing applications.
Where Glyco-Meets-Oligo: OPC Perfected.
At CD Bioglyco, we are committed to providing high-quality Custom Carbohydrate Synthesis services to clients around the world. We provide high-quality OPC-based oligonucleotide fragment purification service to further improve the purity of oligonucleotides by reverse phase chromatography column. The length of oligonucleotides suitable for OPC-based purification needs to be controlled below 35 mer. The purity of oligonucleotides produced by OPC purification is generally guaranteed to be 80-90%. In this purification process, oligonucleotides are prepared on a solid support and then released from the support. The release of oligomers from the support is generally performed by treating the solid support with concentrated aqueous ammonia. It is usually also necessary to remove excess ammonia water under reduced pressure with instruments such as a rotary evaporator.
Specifically, the oligonucleotides synthesized on the supports are treated with ammonia on the respective DNA synthesizers or manually using a syringe. After a period of incubation, collect the ammonia phase. The collected fractions are further deprotected, evaporated, and analyzed to reveal the content of apurinic species in the product. The apurinic site is also cleaved using a mixture of triethylamine and ethanol, with the addition of a small amount of thymidine to quench the released acrylonitrile. Aqueous ammonia is added after cleavage of the oligodeoxynucleotides from the support, and the combined liquid phase is separated from the solid support.
Workflow
Fig.1 General
process of OPC-based oligonucleotide fragment purification. (CD BioGlyco)
Publication Data
DOI.: 10.1021/acs.bioconjchem.0c00717
Journal: Bioconjugate Chemistry
Published: 2021
IF: 3.9
Results: This work details the synthesis of oligonucleotides modified at the 3'-end with amino or thiol groups using novel phosphoramidites derived from 1,2-dideoxy-D-erythro-pentofuranose. A key downstream step involved purifying the synthesized, DMT-protected oligonucleotides after ammonia deprotection. Specifically, the 5'-DMT group was removed using an OPC, which also facilitated desalting. This OPC step provided partially purified, detritylated oligonucleotides containing free amino or protected thiol groups at the 3'-terminus. Final purification to achieve high-purity conjugatable oligonucleotides was then accomplished using reversed-phase HPLC.
Applications
- Oligonucleotides produced by OPC-based purification are used as polymerase chain reaction (PCR) primers.
- Oligonucleotides produced by OPC-based purification are used as primers for DNA sequencing.
- Oligonucleotides produced by OPC-based purification are used as various probes.
Advantages
- By effectively removing truncated and incorrect fragments, our process delivers oligonucleotides with better purity.
- OPC-based oligonucleotide fragment purification is very suitable for synthetic DNA products with a length below 35 mer.
- The purity of oligonucleotides produced by OPC-based purification is generally guaranteed to be 80-90%.
Frequently Asked Questions
Associated Services
OPC-based oligonucleotide purification service delivers rapid removal of salts and small impurities, ideal for high-throughput workflows. To transform these purified fragments into functional tools, our associated services offer customized DNA synthesis solutions:
Precision engineering of modified oligos for probes & diagnostics
High-fidelity long sequences for gene editing applications
Optimized chimeric DNA for next-gen therapeutics
CD BioGlyco has first-class technology and provides clients with comprehensive and reliable Custom Oligonucleotide Synthesis services. We will continue to raise the standard to meet clients' glycobiology research needs. If you are interested in our services, please contact us for more details without any hesitation.
Reference
- Martin-Nieves, V.; et al. Oligonucleotides containing 1-aminomethyl or 1-mercaptomethyl-2-deoxy-D-ribofuranoses: synthesis, purification, characterization, and conjugation with fluorophores and lipids. Bioconjugate Chemistry. 2021, 32(2): 350-366. (Open Access)
