Choosing The Right Method Of Purification For Your Oligos
By Mark Behlke, Integrated DNA Technologies
Many commercial oligonucleotides are deprotected, desalted, quantitated (OD260), and lyophilized before shipping shipping. Most oligos are desalted using a butanol extraction procedure. In some cases, desalting by passage through a Sephadex G25 column is preferred. Either method will remove salt, residual synthesis reagents, and very short oligonucleotide truncation products. Basic desalting-purification is sufficient for routine PCR, sequencing, or probe hybridization applications.
For more demanding applications, such as site directed mutagenesis, cloning, gel shift protein binding assays, etc., additional purification is recommended. We offer preparative scale purification using either denaturing acrylamide/urea gels (PAGE) or reverse-phase HPLC. Added purification will improve the chemical purity of the oligonucleotide and help eliminate undesired reaction products that are less than full length. Truncation of the growing oligo chain during synthesis results in a mix of different species being present in the final product after simple desalting. Truncation products are an unavoidable by-product of any nucleic acid organic synthesis. The relative proportion of undesired truncated material increases as oligo length increases to an extent determined by the efficiency of the synthetic chemistry.
Oligos are synthesized on a solid support matrix one base at a time extending from the 3' terminus. We estimate that each cycle of synthetic chemistry is 98.5 to 99% efficient (Eff = 0.985). The fraction of full-length product present after synthesis will be: (Eff)n-1 or (0.985)19 for a 20-mer, or 75%. For a 50-mer, (0.985) 49 = 48%, for a 65-mer (0.985)64 = 38%, and for a 100-mer (0.985)99 = 22%. Strategies are Inc. into the synthetic chemistry to eliminate undesired truncation products but these are not entirely effective. Thus the final synthesis product after simple desalting must be a heterogeneous mix of desired full-length oligo and undesired truncated products. It is obvious from the above discussion that long oligos should be purified.
We recommend the routine use of PAGE purification for all oligos over 40 bases in length and for shorter oligos whenever the absence of (n-1)-mers is critical for the success of an experiment. In general, PAGE purification provides the best possible enrichment for full-length product. PAGE purification, however, can give low mass yields. HPLC does not remove (n-1)-mer as well as PAGE but has higher yields. We recommend HPLC purification for oligos <10 bases in length and for oligos bearing hydrophobic modifiers that are easily enriched using a reverse-phase approach (see below). Mass recovery usually runs 20-50% for PAGE and 50-70% for HPLC.
Certain oligo modifications couple with lower efficiency than standard bases; enrichment for the desired modified product is beneficial. This is especially true for oligos modified with biotin, digoxigenin, or fluorescent dyes. Purification will increase specific activity and improve signal-to-noise in many applications. Phosphorothioate oligos used in tissue culture or in vivo as antisense agents can be toxic without added purification; we recommend HPLC purification for all phosphorothioate modified oligos.
For more information: Mark Behlke, Vice President, Research and Development Integrated DNA Technologies, 1710 Commercial Park, Coralville, IA 52241. Tel: 319-626-8400, fax: 319-626-8444.