Oligo can be used for targeted gene therapy. It's necessary to analyze and purify the oligo after synthesis to get high purity products for drug discovery or biomedical research.
BOC RNA has built up critical expertise in oligo analysis and purification. In analysis area, we have developed a hexafluoroisopropanol system to analyze the oligo, and can get a better result. In purification area, we have developed RP-IPC to isolate and purify the oligo with high efficiency.
As the high molecule weight (5000-15000), high electronegativity and hydrophobicity of the oligo, we developed a hexafluoroisopropanol system to analyze the oligo due to weak retention and poor peak sharp using traditional RP-HPLC.
Each oligo is coupled sequentially to the growing chain via phosphoramidite method during oligo synthesis. In each coupling cycle, a small amount of the oligonucleotide chains will not undergo base extension, resulting in the accumulation of impurities, these impurities can be failure sequences, shorter sequences, longer sequences, chemically-modified sequences, fluorescence-labeled sequences. These impurities may affect the experiment. Therefore, these impurities must be removed after synthesis.
Then how do you know which purification method is a right choice for you? Your choice of purification should be based on the following factors:
There are some methods for purification
Desalting is a basic method. All of our oligos are desalted.
Cartridge purification removes failure sequences from your final mixture.
The great advantage of RP-HPLC (reversed-phase HPLC) is high resolution, which leads to high purity level. RP-HPLC is an efficient purification method for fluorescence-labeled oligo and large-scale oligo. However, it's not recommended for purifying oligo with more than 50 bases as the purity and yield would reduce.
IE-HPLC (ion-exchange HPLC) is based on the number of charged groups (phosphate) in the oligo backbone. The resolution of this method is very high, but it's not recommended for purifying oligo with more than 40 bases as the purity would reduce.
Due to the low efficiency of IE-HPLC, we developed RP-IPC (reversed-phase ion-pair chromatography) instead to get the oligo isolated and purified with high efficiency.
PAGE is purified based on size and conformation. The purity using PAGE usually can be 95% or more. However, it can only purify a small amount of oligo at a time and the yield is very low.
7. Gel Filtration
Gel filtration is based on molecular weight. The operation is easy and can remove the salts that have been purified by HPLC.
In a word, oligos are best purified by different methods or combinations of methods depending on the application.
With the combined knowledge of the different business units of our parent company BOC Sciences, we are able to help you succeed in your drug discovery and biomedical research.