Q: Why is oligonucleotide purification necessary and how does it affect my experiments?

A: Purification removes incomplete synthesis products and chemical impurities, ensuring specificity and efficiency in applications. Inadequate purification can lead to non-specific binding, reduced efficiency, and inaccurate experimental data.

Inadequate purification can lead to:

• Reduced Specificity: Impurities can interfere with hybridization, leading to non-specific binding or amplification.
• Lower Efficiency: Applications like qPCR or sequencing may yield suboptimal results due to the presence of truncated or impure oligos.
• Inaccurate Data: Contaminants can cause background noise, affecting the reliability of experimental outcomes.
• Toxicity: in vivo experiments may be influenced by toxic byproducts from production or purification.

Therefore, selecting an appropriate purification method is essential to ensure the accuracy and reliability of your experiments.

Purification Methods: Choosing the Right Approach

The best purification method depends on your oligonucleotide’s length and intended application. At Ella Biotech, we offer multiple purification strategies to ensure you get the highest quality oligos:

1. Desalting

Desalting is the most basic purification method, operating by removing small molecular weight impurities and chemical residues, along with truncates shorter than 6 nucleotides. This method is particularly well-suited for shorter oligonucleotides up to 20 nucleotides in length. You’ll find desalting most useful when working with standard PCR primers where achieving the highest purity isn’t your primary concern. However, if you’re doing very sensitive applications like qPCR or if your experiment requires extremely high precision, we recommend to use HPLC purification.

2. RP-HPLC (Reverse-Phase High-Performance Liquid Chromatography)

RP-HPLC is a sophisticated purification method that separates oligonucleotides based on their hydrophobicity. RP-HPLC is particularly valuable for oligonucleotides between 25-50 nucleotides in length. It provides superior purification compared to standard desalting methods. Full-length sequences are typically more hydrophobic than truncated ones, allowing for effective separation. However, please note that the resolution decreases as oligonucleotide length increases, making it less suitable for very long sequences.

At Ella Biotech, we provide RP-HPLC purification free of charge for all modified oligonucleotides. We use this method for oligonucleotides 25 bases or longer, any oligos containing dyes or other hydrophobic modifications, and sequences intended for diagnostic applications.

3. Polyacrylamide Gel Electrophoresis (PAGE)

PAGE is a purification method that separates oligonucleotides based on their molecular weight in a denaturing environment. This technique is particularly effective for very long oligonucleotides (50 bases or longer) or applications requiring the highest purity levels. PAGE offers exceptional single-base resolution and can achieve purity levels exceeding 95% for full-length sequences, making it especially valuable for demanding applications.

While PAGE provides superior resolution and purity for longer sequences, it comes with certain trade-offs. The complex extraction process typically results in lower yields compared to other purification methods. Additionally, the procedure requires pre-purification through HPLC to remove impurities and truncates that could otherwise cause smearing during the PAGE process.

At Ella Biotech, we offer PAGE purification as an optional service, noting that it is a labor-intensive process. Customers should expect approximately 10% lower yield and longer delivery times compared to standard purification methods. For most applications, especially those involving shorter oligonucleotides up to 50 bases, HPLC purification provides a more optimal balance of purity, yield, and efficiency.

4. Ion-Exchange (IEX) HPLC

Ion exchange HPLC, specifically anion exchange chromatography, is a purification method that separates oligonucleotides according to the number of charged phosphate groups, allowing precise removal of deletions and contaminants. This makes it particularly valuable for applications requiring high-resolution separation of shorter sequences, typically less than 20 nucleotides in length.

However, it requires an additional salt removal step after IEX-HPLC purification. This additional step is necessary to ensure that the final product is suitable for downstream applications. While this method provides excellent separation capabilities for short sequences and GC-rich probes, its usage is generally limited to oligonucleotides shorter than 20 nucleotides, making it a more specialized choice compared to other purification methods.

5. Duplex HPLC

Duplex HPLC is a purification method specifically designed for double-stranded oligonucleotides. It uses a second HPLC run on hybridized oligos to get very clean duplexes.

The process uses the fact that single-stranded and double-stranded DNA are very different in hydrophobicity. The second HPLC purification separates the duplexes from excess single strands and mismatched sequences. This process can be done with either reversed-phase or ion-exchange chromatography, depending on the specific characteristics of the oligonucleotides being purified. This purification method is especially useful for applications involving siRNA duplexes and other double-stranded nucleic acids used in research and medical treatments.

6. Reverse-Phase Cartridge Purification

Reverse-Phase Cartridge Purification works through a simple yet effective principle of separating oligonucleotides based on their hydrophobicity, using DMT group binding. It removes many impurities, including some truncated sequences, providing higher purity than desalting. Think of it as a disposable, single-use HPLC column that operates without pressure and requires manual operation. It’s particularly valuable in NGS applications where avoiding cross-contamination between primers is crucial, as the disposable nature of the cartridges eliminates this risk. You can expect to achieve around 70% purity for unlabeled oligonucleotides with this method.

However, this method is not appropriate for high-throughput applications and will result in increased costs.

Q: How should I choose the right purification method for my oligonucleotides?

A: At Ella Biotech, our commitment to quality means we default to RP-HPLC purification for all modified and RNA oligonucleotides including probes. This approach guarantees superior product purity and performance for essential and expensive components. While customers may explicitly request unpurified oligos, we strongly advise against this due to potential risks.

This guide and overview offers recommendations for suitable oligonucleotide purification methods. The choice depends on factors like application sensitivity, oligo length, and modifications. We provide a general purification guide categorized by product type to help you understand the most appropriate method for your specific needs.

1. Primers

For primers, the purification approach depends on length and application sensitivity.

Primers over 30 nucleotides strongly benefit from HPLC purification, while shorter primers often meet requirements through desalting. The key is to match the purification method precisely to the primer’s length and the intended application’s sensitivity.

2. Probes

For probes that typically undergo fluorescence or other modifications, our gold standard is HPLC purification. This method ensures high specificity and minimal background noise, which is critical for applications requiring precise hybridization.

3. TRImers

TRImers demand meticulous purification. Our preferred method is HPLC. In cases requiring additional refinement, we may employ PAGE as an option to achieve maximum precision and minimal impurities.

4. RNA Oligonucleotides

RNA oligos generally require HPLC purification, especially those containing 2’OH groups that cannot be desalted. For antisense oligonucleotides, we treat them similarly to RNA, with HPLC and an added salt exchange to sodium on the DNA backbone as the primary purification method.

5. Modified Oligonucleotides

Modified oligonucleotides sometimes require a tailored approach. For oligonucleotides with fluorescent dye modifications, HPLC is always the method of choice. Other modifications may require varying purification strategies, we can help to determine the most appropriate method for your specific molecular design.