As a biochemist, you’ve doubtlessly encountered the need for using oligonucleotides in various experiments. Designing the perfect oligo for your experiment, however, can be challenging.
The optimal method for oligonucleotide purification depends on three main factors: the length of the oligonucleotide, the pending applications, and the modifications. In this post, we’re going to deal with the best method for the purification of oligonucleotides, as well as the most common techniques used for purification.
What Is Oligonucleotide Purification?
When oligonucleotides are synthesized, they accumulate various impurities – this is unavoidable. Although the appearance of these impurities is in no way strange, they may well jeopardize your experiment. This is precisely what oligonucleotide purification is for.
This process will save you a lot of money in the long run, and prevent heaps of frustration.
The Three Main Factors
We’ve already encountered the three factors of the purification of oligonucleotides. The first one, the length of the oligonucleotide sequence in question is based on one main thing – the longer the sequence is, the more failing sequences will be present.
The second factor, application, essentially asks the question of “What do you need the oligonucleotides for?” As a rule of thumb, going with a purity higher than 95% is advised, just to be safe.
The third factor, modifications, asks the question of “What modifications are you going to be using?” Again, the best way to go is to aim for above-95% purity. Better be safe than sorry.
In essence, the three main factors all dictate oligonucleotide purity of at least 98%.
The Purification Methods
There are fourmajor and widely-used oligonucleotide purification methods: desalting, cartridge purification, PAGE purification, and HPLC purification.Below, we’ll outline each method.
This is the most basic purification process. Using normal phase chromatography, the excess salt from the mixture is removed. The failure sequences, though, remain in place. This lands you with a DNA solution that’s salt-free. Robust techniques, such as ARFP (amplification restriction fragment polymorphism), PCR and microarrays can benefit from desalted oligonucleotide solutions.
With this method, reverse phase chromatography is used. Where desalting removes the salt from the solution and leaves the failure sequences intact, cartridge purification focuses on removing the failure sequences. The end result is a pure material. The product of this method is perfect for cloning, sequencing, preliminary screening, or PCR purposes.
PAGE is short for polyacrylamide gel electrophoresis. On the basis of conformation and size, the PAGE method differentiates the sequence failures and the desired sequence. While it’s comprehensive and effective, the downside of using this method is that small oligo amounts end uppurified each time. The yield is also abysmal. The end result, however, is an oligo with a purity of at least 95%. The PAGE method is used in gene synthesis, X-ray crystallography, as well as in mutagenesis research.
Short for high-pressure liquid chromatography, HPLC, like PAGE and cartridge purification, also focuses on failure sequences removal. Highly-purified oligonucleotides are pretty much guaranteed with HPLC. Compared to the above-mentioned methods, HPLC delivers high purity and can handle large oligonucleotide amounts. However, this technique is complex and requires the use of expensive equipment. HPLC purification is predominantly used in a diagnostic’s context.
Which One to Go with?
Each of the mentioned methods has its applications. Sometimes, you don’t need the mixture to be completely clear of all failure sequences. Other times, purity is absolutely necessary.
Basically, ask yourself how many purified oligonucleotides are we talking about here? How pure does the mixture really need to be for your needs? Combine these questions with the factors outlined before, and you should have the answer as to which oligonucleotide purification method to go with.
If you are still unsure what to do, take a look at various scientific papers available online. If you have questions, go ahead and send a couple of emails to the paper authors. More than likely, they’ll be happy to help.
Choosing the Perfect Method
As you can see, things aren’t black and white here. There are various applications towards purifying oligonucleotides. The best way to go is to aim at least 95% purity at all times. Furthermore, do your research – go ahead and read the research paper available online. You have nothing to lose and only knowledge to gain.