Who amongst us hasn’t had the need for oligonucleotides in an experiment? It is a cornerstone in many procedures and techniques. Depending on the goal, it can be very hard to design just the right oligo for your experiment. Oligos must have the right length; the right amount of C-G, T-A; they can’t form secondary structures; they must have the proper oligo purification process, etc.
But, worry not. We are here to give you a helping hand.
What Is the Oligo Purification Process?
Oligonucleotide synthesis leads to the inevitable accumulation of impurities. These impurities are failure sequences: shorter sequences, longer sequences, modified sequences; or impurities left over by the synthetization process. These impurities may jeopardize your experiment, as they compete with the full-length product in some applications, or inhibit reactions. Therefore, the purification process saves you money in the long run… and also a bit of your sanity!
So, now the big question…
How Do I Know Which Purification Process Is Right for Me?
Well, that is the big question, as there are many different techniques to purify the oligonucleotides. The decision mostly relies on the amount to be purified, and the degree of purification required for the technique.
You are probably thinking: Well, I need my oligonucleotides to be as pure as possible, obviously. And you are right, to a certain degree. While we want everything to be as pure as possible, we know that there are some robust techniques that can handle a bit of impurities in the mix – without compromising our experiment, results, sanity and wallet.
Therefore, it is important to know exactly what is the role of your oligonucleotides in your experiment, and just how robust the technique is. And then, you just have to choose:
Desalting is a very basic process of purification. Excess salt is removed from your mixture using normal phase chromatography. It does not remove failure sequences. It yields a salt-free, ready to use DNA-solution, great for robust techniques, like PCR, amplification restriction fragment polymorphism (ARFP) analysis, microarrays, etc.
You can also use desalting in combination with other purification techniques!
Cartridge purification uses reverse phase chromatography. Unlike desalting, cartridge purification removes failure sequences from your final mixture. This method results in a fairly pure material and the product is great for sequencing, cloning, PCR or preliminary screening purposes.
The poly acrylamide gel electrophoresis (PAGE) method of purification differentiates between the desired sequence and sequence failures based on size and conformation. The disadvantage of this method is that only very small amounts of oligo are purified at a time and the yield is quite poor; however, it results in an oligo with purity greater than 95%. PAGE purified oligos are often used for X-ray crystallography, gene synthesis, and mutagenesis studies.
High pressure liquid chromatography (HPLC) also removes failure sequences from your mixture – and guarantees highly purified oligonucleotides. This purification technique purifies large amounts of oligonucleotide at high purity, which represents a strong advantage over the methods mentioned above. HPLC is, therefore, a great purification tool for acquiring large amounts of highly purified oligonucleotides – usually needed in a diagnostic’s context.
Yeah, but How Do I Know Which One to Use?
Your choice is based two things: the amount of purified oligonucleotides you need, and how pure does it needs to be. Now this may feel a bit empirical, however always keep in mind what your goal is, and how robust is your technique.
If you are still not sure which one to choose, seek help from scientific papers that have used the technique you want to use: see which purification method they chose and how it worked out for them. When in doubt, don’t hesitate to send them an email with your questions – after all, Science is all about sharing knowledge. You can also ask the company responsible for the purification process. They have extremely qualified professionals that can help you out.
As long as you know what you want to accomplish with your experiment, and how robust your technique is, you should be alright. If not, seek out opinions and advice. If everything else fails, go for the old: “Let’s try this one out, and see how it works!”Image credit: sabertasche2