Sequence-specific purification of DNA oligomers in hydrophobic interaction chromatography using peptide nucleic acid amphiphiles: extended dynamic range.
We present improvements on a previously reported method (Vernille JP, Schneider JW. 2004. Biotechnol Prog 20(6):1776-1782) to purify DNA oligomers by attachment of peptide nucleic acid amphiphiles (PNAA) to particular sequences on the oligomers, followed by their separation from unbound oligomers using hydrophobic interaction chromatography (HIC). Use of alkyl-modified HIC media (butyl and octyl sepharose) over phenyl-modified media (phenyl sepharose) reduced the elution time of unbound DNA while not affecting the elution time of the PNAA/DNA complex. Modifying the alkane tail length for PNAA from C(12) to C(18) increased slightly the retention of PNAA/DNA duplexes. By combining these two refinements, we show that sequence-specific purifications of DNA oligomers 60 bases in length or more can be achieved with high resolution, even when the PNAA alkane is attached to the center of the target strand. The insensitivity of the PNAA/DNA duplex binding to choice of HIC media appears to be due to a surface-induced aggregation phenomenon that does not occur in the case of untagged DNA. We also report on the use of batch HIC as an adequate predictor of elution profiles in linear gradient HIC, and its potential to considerably reduce purification times by applying step gradients.
Savard
Jeffrey M
JM
Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213-3890, USA.
Schneider
James W
JW
D002845:Chromatography; D002847:Chromatography, Agarose / Q000379:methods*; D004247:DNA / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D009841:Oligonucleotides / Q000737:chemistry* / Q000302:isolation & purification*; D020135:Peptide Nucleic Acids / Q000737:chemistry*
D009841:Oligonucleotides; D020135:Peptide Nucleic Acids; D004247:DNA