Oligonucleotide Purity Analysis
Oligonucleotides are typically composed of short chains of nucleotides (deoxyribonucleotides or ribonucleotides) of 20 or fewer and are short RNA and DNA oligomers. They can......be used as probes to determine the structure of DNA or RNA, and are utilized in processes such as gene chips, electrophoresis, and fluorescence in situ hybridization....
Oligonucleotides are mainly synthesized chemically and are widely used in fields such as biology, agriculture, and medicine. Currently, automated oligonucleotide synthesizers have been developed that can rapidly synthesize oligonucleotide fragments and fluorescently labeled oligonucleotides of various sequences. During this process, impurities can be introduced at each step, including unreacted raw materials, reaction by-products, degradation products, and oligonucleotides of different sequences...etc. These impurities not only cause inaccuracies in the quantification of oligonucleotides but may also affect subsequent biological experiments. Therefore, synthesized oligonucleotides need to be purified to ensure the accuracy of subsequent experiments.
Currently, oligonucleotide purification methods are roughly divided into two types: one is the conventional method of controlling the quality and length of oligonucleotides for separation, and the other involves specific adsorption of the 5′-hydroxyl incomplete dimethoxytrityl (DMT) protecting group......
(1) Control of oligonucleotide quality and length: Existing methods for controlling the quality and length of oligonucleotides include polyacrylamide gel electrophoresis (PAGE) and high-performance liquid chromatography (HPLC). Both methods are based on the difference in migration speed of oligonucleotides of different lengths in the medium to separate the target oligonucleotide, but the throughput is generally low.
PAGE: Due to differences in charge and size of molecules, their migration speed in the gel is affected, with larger fragments migrating slower. After electrophoresis for a certain period, fragments of different sizes can be separated.
Characteristics: Complex operation, long analysis time, and difficulty in achieving automated separation and analysis; high resolution and no restriction on molecular length.
HPLC: HPLC is currently the main method for the separation and purification of oligonucleotides. The separation and purification criteria are the separation degree of the target and impurities. The existing HPLC method uses TEAB as an ion-pairing reagent for the separation and purification of oligonucleotides, known as the reverse-phase ion-pair (IP-RP) chromatography method.
Characteristics: High degree of automation, labor-saving, high product purity; however, the purification quantity is small, speed is slow, cost is high, and efficiency in batch production is low. Additionally, due to the memory effect of the chromatographic column, the possibility of cross-contamination should also be considered.
(2) Specific adsorption based on DMT groups: Target oligonucleotides can selectively carry a DMT group after the synthesis of the last base, while prematurely terminated oligonucleotides do not carry this group. This difference can be recognized by media that specifically bind to DMT groups, thereby only collecting the target oligonucleotide. This method has a relatively high throughput but is only suitable for shorter oligonucleotides, and an additional step to remove the DMT group is required after purification.
OPC column purification: Utilizes the affinity between the column material and the 5′-end DMT of the synthesized primer to specifically adsorb DMT-containing oligonucleotide molecules. Therefore, in subsequent processing after synthesis, care should be taken not to remove the DMT group, and this group should be removed during the purification process.
Characteristics: Efficient at removing salts and short fragments, and the purified product can achieve a high purity suitable for most molecular biology experiments. This method is fast, efficient, and can be automated. However, it is limited by length and column capacity, and the purification degree is limited.
(3)Desalting purification:For oligonucleotides without a DMT protecting group, solutions of oligonucleotides collected after electrophoresis separation and anion exchange liquid chromatography purification need further desalting. If the synthesized oligonucleotide quality is very high, direct desalting purification, which only removes non-DNA impurities, can be used without significantly affecting downstream operations. Solid-phase extraction (SPE) technology is generally used for desalting....
SPE:The principle is that oligonucleotides can be specifically adsorbed onto a reverse-phase adsorbent, can be eluted by organic solvents but not by water, thereby effectively removing salts.
Characteristics: Suitable for oligonucleotides with relatively high purity, effectively removes salts, cannot remove fragments, and is a quick and simple desalting method.
Oligonucleotide purity analysis is an important aspect in the field of biomedicine and plays a key role in ensuring the quality and safety of oligonucleotides. With the continuous development and innovation of biotechnology, oligonucleotide purity analysis methods are also constantly being improved and optimized. BTP BioTech, based on a high-resolution liquid chromatography platform, has developed and validated oligonucleotide purity analysis methods based on purification analysis principles. These methods can select suitable column lengths, mobile phase flow rates, separation temperatures, and ion-pairing reagent buffer components according to different oligonucleotide samples to identify impurities such as n-1 short-chain bodies, n+1 long polymers, and phosphodiesters resulting from incomplete sulfurization and side reactions due to reaction failures. For more oligonucleotide services, feel free to consult us for free!
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