Length-dependent DNA separations using multiple end-attached peptide nucleic acid amphiphiles in micellar electrokinetic chromatography.

End-labeled free-solution electrophoresis (ELFSE) is an alternative approach to gel-based methods for size-based electrophoretic separation of DNA. In ELFSE, an electrically neutral "drag-tag" is appended to DNA to add significant hydrodynamic drag, thereby breaking its constant charge-to-friction ratio. Current drag-tag architecture relies on covalent attachment of polymers to each DNA molecule. We have recently proposed the use of micellar drag-tags in conjunction with sequence-specific hybridization of peptide nucleic acid amphiphiles (PNAAs). This work investigates the effect of multiple PNAA attachment on DNA resolution using MEKC. Simultaneous PNAA hybridization allows for the separation of long DNA targets, up to 1012 bases, using micellar drag-tags. Each PNAA handle independently interacts with the micellar phase, reducing the overall mobility of this complex relative to individual PNAA binding. The sequence- and size-based dependence of this separation technique is maintained with multiple PNAA binding over a range of DNA sizes. Results are accurately described by ELFSE theory, yielding alpha=54 for single-micelle tagging and alpha=142 for dual-micelle tagging. This method is the first example of a non-covalent drag-tag used to separate DNA of 1000 bases based on both size and sequence.

Identification of PCR products using PNA amphiphiles in micellar electrokinetic chromatography.

We present a method to identify single-stranded PCR products of varying lengths by hybridization of n-alkylated peptide nucleic acids (PNA amphiphiles) to the products, followed by separation with micellar electrokinetic chromatography (MEKC). These end-attached PNA amphiphiles (PNAA) partition to nonionic micelles in the running buffer (Triton X-100), linking the tagged DNA to the micellar drag-tag. This linkage shifts the electrophoretic mobility of a tagged component away from both untagged DNA and tagged DNA of different lengths. The mobility of the tagged DNA is established by its extent of partitioning to the micelle phase as well as its size relative to the attached micelle. A model is presented that can be used to determine the length of an unknown oligomer given an experimentally obtained mobility. We find that the collective action of micelles that transiently attach to the tagged DNA impart about the same hydrodynamic drag as covalently bound "drag-tags" of a similar size. With the use of the PNAA-MEKC method, PCR products of 88, 134, 216, and 447 bases are clearly resolved in less than 5 min. To our knowledge, this work represents the first use of surfactant micelles as drag-tags to separate DNA in capillary electrophoresis. Furthermore, the PNAA tag only attaches to DNA containing a target sequence, helping ensure that only the desired PCR products are analyzed.

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.