Oligonucleotide
Quality Control

A: At Ella Biotech, ESI- or MALDI-MS analysis is standard procedure for ensuring the quality of all modified and RNA oligos. The same techniques are used for statistical checks on primers and unmodified oligos. This quality control method not only guarantees client satisfaction without significantly impacting delivery schedules, but it also strengthens our assurance in providing accurate, superior oligonucleotides.

We use several complementary techniques, each offering unique insights into different aspects of oligonucleotide quality.
The following are our primary methods of analysis:

Mass Spectrometry Methods

For most routine quality control, we primarily use two types of mass spectrometry:

MALDI-MS (Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry)
MALDI-MS is a soft ionization technique that allows the analysis of oligonucleotides by ionizing them without significant fragmentation. This is our method of choice for high-throughput quality control. We check oligonucleotides of each production run as well as all produced modified oligos and dual-labelled probes. It works particularly well for oligonucleotides up to 40 bases long and gives us rapid results that confirm whether we’ve synthesized exactly what you ordered.

ESI-MS (Electrospray Ionization Mass Spectrometry)
When dealing with longer sequences (particularly above 30 bases), we turn to ESI-MS. It works by ionizing a small sample of the oligonucleotide, giving it multiple negative charges. These charged molecules are then sent to a mass analyzer for detection. 
The raw data shows multiple peaks, each representing a differently charged version of the molecule. A special deconvolution algorithm is used to combine all these peaks and calculate the actual mass of the oligonucleotide. This measured mass is then compared to the theoretical mass based on the sequence to confirm that the oligonucleotide has been correctly synthesized.

A key advantage of ESI-MS is that it does not break or fragment the oligonucleotide during ionization, making it ideal for analyzing longer sequences, up to about 150 bases. Though it requires more careful sample preparation.

Chromatography Methods

We use two main types of chromatography, each with its own strengths:

Reversed-Phase HPLC (RP-HPLC)
This method separates oligonucleotides based on their hydrophobicity. Full-length sequences (with hydrophobic trityl groups) are retained longer than shorter sequences . Thus, oligos and their impurities elute at different times due to variations in hydrophobic interactions. RP-HPLC is excellent for both purification and analysis of oligo products.
It’s especially good for oligonucleotides up to 50 bases, can be used up to 80 bases, but somehow less effective.

Ion-Exchange HPLC (IEX-HPLC)
IEX-HPLC separates oligos based on their electrical charge differences, which correlate with the number of phosphate groups in the backbone. Anionic oligos interact with a cationic stationary phase, and elution is achieved by increasing the ionic strength of the mobile phase. It’s particularly effective for oligos up to 40 bases and works especially well with sequences that have high GC content or secondary structure. Consider it our specialist tool for handling tricky sequences that might be challenging to analyze by other methods.

PAGE (Polyacrylamide Gel Electrophoresis)

PAGE separates oligos based on their size and conformation by applying an electric field through a polyacrylamide gel matrix. At Ella Biotech, we use PAGE mainly for quality control as well as for purification on customers´ request.
We recommend PAGE analysis and purification for long oligos, when other methods may not work well in these cases. PAGE purification usually makes oligonucleotides very pure, but this process can reduce the amount of product. The final analysis of dye-labeled oligonucleotides with PAGE is done on a fluorescent scanner to identify any potential side products.