28 May / June 2017
A poly-dT 12-40mer was separated at high temperature and high flow rate with a 4-minute gradient using triethylammonium acetate (TEAA) as mobile phase. In addition, up to 60mer poly-dT oligonucleotide was separated using a 7-minute gradient. In both cases, a convex gradient was used to maximise the resolution of the longer oligonucleotides in a shorter time.
Separation of Failure Sequences
Among the most common impurities in synthetic oligonucleotides are failure sequences. While each coupling step is highly efficient, the probability of coupling failure increases with each step. Hence, one or two nucleotide ‘deletions’ can be expected in most synthetic oligonucleotides. Figure 3 shows the separation of a 25mer DNA from its 5’ deletion sequences (n-1 and n-2).
Separation of Fluorescent Dye-Labelled DNA
Fluorescent dye-labelled oligonucleotides are used in many applications such as DNA sequencing, PCR, DNA microarrays and in situ hybridisation. In most cases, attachment of a fluorophore increases the overall hydrophobicity of the oligonucleotide sample, resulting in stronger retention on a reversed-phase stationary phase. Figure 4 shows the separation of a fluorescein labelled oligonucleotide and its unmodified form. The unmodified oligonucleotide and other impurities were separated from the main dye-labelled oligonucleotide peak.
Separation of Large Double- Stranded DNA Fragments
Purification and sizing of large double- stranded DNA fragments is an important step in DNA cloning, PCR and for the preparation of DNA libraries for HTS or NGS. Traditionally, these dsDNA fragments are separated by agarose or acrylamide gels, and extracted from the gels for subsequent use. This process requires manual excision of the target DNA from the gel, and extraction of the DNA from the excised gel. This laborious and time consuming step generally produces yields of less than 50%. The DNAPac RP provides reliable separation and higher yields and DNA fragment collection is readily automated. Many reversed-phase columns advertised for such separations harbour small pores which restrict separation of dsDNA fragments over 600-1000 base pairs (bp). The wide- pore DNAPac RP resin produces resolution
Figure 4. Separation of Fluorescein-Labelled DNA.
Figure 5. Separation of dsDNA fragments , showing a restriction enzyme digest and a DNA ladder. [2].
comparable to many DNA gels up to 10K bp, and in similar times. Figure 5 shows the separation of dsDNA fragments generated from a restriction enzyme (bottom panel) and a DNA ladder (top panel). Fragments ranging from 72 bp to 10,000 bp were separated on the DNAPac RP within 15 minutes [6].
Summary
The therapeutic use of oligonucleotides will likely arrive at the markets within the next few years if all goes well with current phase 3 trials, offering hope to those whom these therapies seek to help. With the complexity and analytical challenges that
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