Oligonucleotides are assembled via solid-phase synthesis. In that way, nucleotides are attached according to the sequence of choice leading to chain growth. It is generally accepted that chemical reactions never reach 100% conversion. However, in state-of-the-art oligonucleotide synthesis, coupling yields of up to 99.5% are achieved. With the unreacted 0.5% of the strands, chain assembly stops and truncated sequences form as side products. Depending on the intended application, it is advantageous to remove these shorter sequences from the full-length product.

RP-HPLC Purification

Oligos <50 nt in length can be well purified via RP-HPLC (reverse-phase high-performance liquid chromatography). Through this purification approach, preferably residual, n-x truncated oligos (lacking the hydrophobic DMT protection group at the 5’ end) are removed. This results in a ≥85% purity of the targeted oligonucleotide. RP-HPLC is useful for a higher level of purity that is required for more demanding applications, such as cloning, DNA fingerprinting, real-time PCR, FISH, etc.

Potential Applications:

Molecular cloning

DNA fingerprinting

Real-Time PCR and digital PCR*

FISH

* The primer and qPCR probes are compatible with all commercially available supermixes

IEX-HPLC Purification

IEX-HPLC (ion-exchange high-performance liquid chromatography) is a preferred purification method for longer oligonucleotides (40-80 nt). Through this purification approach, residual n-x truncated oligos are removed in an efficient way. Whereas RP-purification yields very good results for oligos < 50 nt, IEX purification is superior for longer oligonucleotides. (40-80 nt). For oligos with this length, IEX results in ≥85% purity of the targeted oligonucleotide. IEX is useful for a higher level of purity of long oligonucleotides required for more demanding applications, such as direct cloning or NGS applications.

Potential Applications:

Molecular cloning (direct cloning)

NGS Applications

HPLC Purification & Dialysis

Dialysis as an add-on to HPLC is recommended if oligos need to be present in a physiological state. This purity level is strongly recommended when performing in vivo experiments (e.g. in mice).

Potential Applications:

Antisense experiments

Cell culture studies

Physical Chemistry and Structure Analysis (NMR, MS, etc.)