(366b) Sequence Specific Separation of Target DNA in Micellar Electrokinetic Chromatography

We present the use of hydrophobically labeled probes for the sequence-specific separation of oligomeric DNA from complex mixtures using micellar electrokinetic chromatography (MEKC). DNA hybridization and micelle interaction are achieved using a peptide nucleic acid (PNA) appended to an alkane tail, forming a peptide nucleic acid amphiphile (PNAA). Uncharged PNAAs bind DNA oligomers with high sequence-specificity, tagging them for separation by either their hindered electrophoretic mobility or their partitioning to surfactant micelles in the running buffer. In the absence of micelles, PNAA attachment to short DNA oligomers is sufficient to alter the electrophoretic mobility, providing good resolving power between target and non-target DNA. However, targeting sequences greater than 20 bp in length becomes problematic as the impact of PNAA attachment on the mass-to-charge ratio of the PNAA/DNA duplex becomes progressively small.

MEKC offers an additional mechanism for separation; that is, the partitioning of hydrophobically labeled DNA to micelles present throughout the running buffer. Facile separations of 60 bp DNA are achieved using running buffers containing Triton X-100 at concentrations above its CMC. The use of an excess of surfactant required for MEKC may also improve the compatibility of the process with contaminated samples, as lipophilic material is solubilized in the micelle interior rather than adsorbing to the capillary wall. We will discuss efforts to multiplex the sequence-specific separation method by using multiple PNAAs with varying alkane tail lengths. PNAA with longer alkane chains partition more strongly to the micelle pseudophase so that distinct sequences are discriminated. Finally, we will discuss efforts to miniaturize the method for microscale bioanalytical devices.