Ready-to-use primers and probes in a single purification step
In-house synthesized primers and probes may contain traces of salts or dyes that inhibit downstream applications. In particular, probes suffer from unbound fluorescent dyes that interfere with signal detection and lead to erroneous PCR results.
Kilobasers new OliPure HIC kit easily removes these contaminants in a single spin.
Why do we need to purify oligonucleotides?
Oligonucleotide synthesis inevitably leads to the accumulation of impurities that can jeopardize downstream applications. Small molecule impurities are a by-product of the cleavage and deprotection process and increase in concentration with the length of the oligonucleotide. These small molecules are hydrophobic and inhibit enzymes by binding to the substrate binding site.
When labeling oligonucleotides, such as probes, with fluorescent dyes, unbound dyes may remain with the final product. These unbound dyes produce background noise that can seriously interfere with signal detection. In the case of dual-labeled probes for PCR, this may lead to erroneous results.
Therefore, the functionality of oligonucleotides can be significantly improved by the proper purification technique.
How does the OliPure HIC Purification Kit work?
HIC stands for Hydrophobic Interaction Chromatography, which is used to separate proteins based on their hydrophobicity. The HIC column can be built with various hydrophobic modified materials. Our material of choice is phenyl-modified agarose. It combines the property of an aromatic ring with the property of agarose. Using phenyl-agarose as our purification column, small molecule impurities and unbound dyes are retained as follows.
Removal of salts
Agarose is commonly used in size exclusion chromatography (SEC) for the separation by size of small molecule compounds. The size, or more precisely, the hydrodynamic volume, is measured by how efficiently these compounds penetrate the pores of the stationary phase.
The agarose forms a porous matrix with a range of different pore sizes. Smaller molecules can easily move in and out of the pores, while larger molecules, complexes and particles are too large to enter the pores. As a result, large molecules leave before small molecules do.
Figure 1: Principle of size exclusion chromatography: Molecules penetrate the pores of the agarose depending on their size. Since small molecules move into the pores more easily than large molecules, large molecules will leave the agarose-filled column before small molecules do.
In case of oligonucleotide synthesis, small molecule impurities are typically inorganic salt ions of a small size (< 2 Å); they penetrate the small pores present in the agarose mand are consequently retained in the column. The much larger oligonucleotides (50 Å), however, cannot enter the pores and leave the column immediately.
In this way, the OliPure HIC Purification Kit removes all residual by-products from the synthesis, cleavage, and deprotection processes.
Removal of fluorescent dyes
For a molecule to be fluorescent, certain molecular structures are required, such as an aromatic ring system. The most commonly used fluorescent dyes in molecular biology—FAM, HEX, and TET—are all based on fluorescein, an organic dye based on the xanthene motif with three aromatic rings.
Aromatic rings have a unique noncovalent binding property called pi or π-π stacking. Through the use of phenyl-modified agarose, the phenyl groups are present on the inside of the column. They bind unbound dyes via pi stacking so strongly that they remain in the column almost permanently. In contrast, dyes attached to the oligonucleotide cannot reach the phenyl groups inside the column due to the size and polarity of the oligonucleotide. Therefore, labeled oligonucleotides are not affected by the column and leave the column immediately.
In this way, the OliPure HIC Purification Kit removes all unbound dyes.
Figure 2: Binding of dyes within the column: Most fluorescent dyes (in green) consist of aromatic rings that are bound by noncovalent interactions with phenyl groups (in dark blue) when they enter the pores of agarose (in light blue).
What makes this one different from other purification kits?
Like many other systems for purifying DNA/RNA strands, the HIC column is placed in a spin tube to allow purification by centrifuging. However, the OliPure HIC purification kit is based on a different principle of purification than most other systems.
In many commonly used systems, DNA/RNA strands are first bound and then eluted in a separate step, requiring multiple solvent exchanges before the purified oligonucleotide is obtained. What is more, binding efficiency increases with length and hence, shorter oligos bind less efficiently, resulting in higher losses and low recovery rates for oligonucleotides shorter than 50 nt.
The OliPure HIC Purification Kit, by contrast, is optimized for the purification of small oligonucleotides (>12nt). This is made possible by basing our purification method not on binding, but on affinity to the column. Since oligonucleotides are not retained by the column (as described above), purified DNA/RNA strands can be obtained in a single spin-down.
Thanks to the reduction of overall steps, the OliPure HIC Purification Kit purifies DNA/RNA oligonucleotides in less than 10 minutes.
What can you do with it?
The OliPure HIC Kit is the optimal complement to our small scale DNA/RNA synthesizer. It allows for the purification of all oligonucleotides that can be synthesized with Kilobaser one and one-XT. Additionally, it can easily be adapted to purify oligonucleotides of various other sources.
Based on the current features of the kit, its main functions are as follows:
- Purification of probes after synthesis
- Desalting of oligonucleotides after synthesis
- Purification of probes after fluorescent labeling
- Removal of phenol after phenol-chloroform extraction
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