Necessity of Oligonucleotide Fragment Purification

Oligonucleotides, a term denoting short nucleotide chains with less than 20 bases, possess an innate propensity for facile hybridization with their complementary counterparts. This feature underpins their common utility as molecular probes, finding application in elucidating the structural characteristics of DNA or RNA, evident in techniques such as gene chips, electrophoresis, and fluorescence in situ hybridization. Yet, the process of designing an accurate oligonucleotide sequence can be intricate. The synthesis of oligonucleotides frequently engenders the inadvertent emergence of impurities. These impurities bear the potential to perturb experimental outcomes, either through competitive interaction with the intended full-length product or by constraining the desired reactions. Consequently, the implementation of purification protocols becomes indispensable to ensure the fidelity of oligonucleotide samples intended for experimental deployment.

Key Technologies

Reversed-phase high-performance liquid chromatography (RP-HPLC); Anion-exchange HPLC (AE-HPLC); Polyacrylamide gel electrophoresis (PAGE)

Where Every Fragment Meets Its Perfect Match.

Custom Carbohydrate Synthesis is one of the key services we have been focusing on. CD BioGlyco with many chemists offers advanced custom carbohydrate synthesis for global clients. At CD BioGlyco, we provide oligonucleotide fragment purification services through multiple advanced technologies, some of which are shown below.

• Desalination-based oligonucleotide fragment purification
  Desalting stands as a fundamental purification technique employing both normal phase and reverse phase chromatography. Its primary purpose is the removal of surplus salts from a mixture, yielding a salt-free DNA solution that is readily applicable. The resulting purified oligosaccharides adhere to the prerequisites of various experiments, including applications like microarrays and more.
• Oligonucleotide purification cartridge (OPC)-based oligonucleotide fragment purification service
  The OPC column is equipped with a resin that has an affinity for DMT. When synthesizing DNA fragments, the DMT on the last base at the 5' end is retained. After all the synthetic products are adsorbed on the OPC column, wash the column with a dilute organic solvent. Fragments have strong adsorption ability and are not easy to be eluted. Fragments without DMT have weak adsorption ability and are eluted. Then trifluoroacetic acid or trichloroacetic acid is used to remove the DMT group, and then elute the DNA by a concentrated organic solvent. This method has the characteristics of fast and easy.
• HPLC-based oligonucleotide fragment purification service
  This approach is well-suited for experiments demanding elevated oligonucleotide purity, encompassing applications like directed mutagenesis, quantitative gene detection, and diagnostic assays. For the purification of oligonucleotides, either anion exchange resins or reverse resins are employed. High-performance liquid chromatography (HPLC) conducted with anion exchange resins routinely attains purification efficiencies in the range of 95-98%. This method is marked by its substantial degree of automation, swiftness, and the attainment of elevated product purity levels.
• PAGE-based oligonucleotide fragment purification service
  PAGE purification is to distinguish the target sequence from the failure sequence according to the size and conformation. The technique utilizes the different charges and electrophoretic mobility of long and short fragments to separate them. Purified oligosaccharides produced by this technique are commonly used in X-ray crystallography, gene synthesis, and mutagenesis studies.

Workflow

Our purification process is a rigorous, multi-stage workflow tailored to each project's unique requirements to ensure the highest possible quality.

• Crude Oligonucleotide Receipt and Desalting

The process begins with the receipt of the crude oligonucleotide synthesis product. The initial step is a comprehensive desalting procedure to remove small molecular weight impurities, such as synthesis byproducts and salts, which can interfere with subsequent purification steps.

• Primary Purification

The desalted product undergoes a primary purification step using a chosen advanced technology, such as RP-HPLC. The trityl-on method is often used here, allowing us to specifically isolate the full-length oligonucleotide from shorter, truncated sequences.

• Final Detritylation and Isolation

After primary purification, the protective trityl group is removed from the isolated full-length fragment. The product is then meticulously collected and prepared for the next stage.

• Secondary Purification and Quality Control

Depending on the purity required, a second purification step, such as AE-HPLC, may be performed to ensure the highest-grade product. The final purified oligonucleotide is then subjected to a battery of quality control tests, including mass spectrometry to verify molecular weight and analytical HPLC to confirm purity.

Publication Data

Applications

• Genetic research: Purified oligonucleotide fragments are essential for genetic research, including PCR, DNA sequencing, and mutation analysis. Accurate and reliable fragment purification ensures the integrity of genetic information and enhances the accuracy of experimental outcomes.
• Gene editing and CRISPR-Cas9 technology: In gene editing applications like CRISPR-Cas9, precise purification of oligonucleotide fragments ensures the accuracy of gene modification. Purified fragments are used as guides or templates for targeted gene editing processes.
• Diagnostic assays: Purified oligonucleotide fragments play a key role in diagnostic assays, such as nucleic acid-based tests for the detection of pathogens, genetic markers, and mutations. Highly purified fragments are essential to avoid false positives or negatives.
• Functional genomics: Oligonucleotide fragments are used to study gene expression, protein-DNA interactions, and other molecular mechanisms. Purified fragments are critical for obtaining reliable and reproducible results in functional genomics experiments.

Advantages

• A professional Glyco™ Synthesis Platform.
• Improved sensitivity: Highly purified oligonucleotide fragments increase the sensitivity of diagnostic assays and research experiments. Minimizing background noise and interference enhances the detection of target sequences or interactions.
• Facilitates therapeutic development: For applications in drug discovery and development, such as antisense oligonucleotide therapeutics, the purity of oligonucleotide fragments is crucial for safety and efficacy. Purification ensures the removal of potential contaminants that could lead to adverse effects.
• Time and cost savings: Outsourcing oligonucleotide fragment purification to specialized services saves time and resources.

Frequently Asked Questions

Associated Services

To unlock advanced functionality building upon this high-purity foundation, our associated services enable site-specific base modifications, including:

At CD BioGlyco, our Custom Oligonucleotide Synthesis service ensures the precise design and construction of oligonucleotides tailored to your specific research needs. Utilizing cutting-edge technologies, our experts purify high-quality oligonucleotides, enabling you to advance your research with confidence. If you are interested in our services, please do not hesitate to contact us for more details.