Purification of oligonucleotides

The lipophilicity of this phosphate protective group helps in the chromatographic purification of oligonucleotides. It is removed by the oximate method. ... 

This highly lipophilic group is cleaved with isoamyl nitrite in Pyr/AcOH. The use of a lipophilic 5 -phosphate protective group aids in reverse-phase HPLC purification of oligonucleotides. 

Gerstner, J. A., Economics of displacement chromatography — a case study purification of oligonucleotides, BioPharm, 9, 30, 1996. 

Farmer, J.C., and Castaneda, M. (1991) An improved preparation and purification of oligonucleotide-alkaline phosphatase conjugates. Bio. Tech. 11, 588-589. 

TABLE I Application of Various Chromatographic Techniques for Purification of Oligonucleotides and Nucleic Acids... 

Agrawal, S., Habus, I., and Kandimalla, E. (1998). Affinity-based purification of oligonucleotides using soluble multimeric oligonucleotide. PCT Int. Appl. WO 98/04571. 

Johansson, H. J., and Svensson, M. A. (1995). A novel method for large-scale purification of oligonucleotides. Nucleic-acid Based Thera., San Diego, CA., 1995. 

A wide range of techniques is available for the separation and purification of oligonucleotides after the removal of protecting groups. Obviously, chemical synthesis is not the only source of such compounds, and those derived by purely enzymic syntheses, or after isolation from biological sources, have been examined in similar ways. The literature is correspondingly extensive, and only those methods routinely used after a chemical synthesis will be discussed in detail here. 

Cohen AS, Najarian DR, Paulus A, Guttman A, Smith JA, Karger BL (1988) Rapid separation and purification of oligonucleotides by high-performance capillary gel electrophoresis. Proc Natl Acad Sci USA 85 9660-9663. 

In addition, fluorous tags have been developed for purification of oligonucleotides [18] and oilgosaccahrides [19] synthesized on solid support. 

Figure 36. Biotin-avidin system for detection and purification of oligonucleotides. Modified from [277] Examples for direct ligand incorporation.

If oligodT was used for cDNA synthesis, then add the arbitrary primer at this point. The optimal primer concentration in the PCR reaction ranges from 0.3-10 M. This parameter will depend on the primer sequence and on the particular primer preparation used. To minimize effects of the latter, purification of oligonucleotides through denaturing aaylamide gels is recommended. In a typical reaction, we use the primer at 1 xM. 

Release and purification of oligonucleotide as in phosphoramidite synthetic route... 

Gerstner JA. Economics of displacement chromatography—A case study Purification of oligonucleotides. Biopharm 1996 9 30-36. 

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