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.
Grosser
Shane T
ST
Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213-3890, USA.
Savard
Jeffrey M
JM
Schneider
James W
JW
D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods*; D004247:DNA / Q000302:isolation & purification*; D008970:Molecular Weight; D020135:Peptide Nucleic Acids; D016133:Polymerase Chain Reaction; D013501:Surface-Active Agents; D047071:beta-Glucans
D020135:Peptide Nucleic Acids; D013501:Surface-Active Agents; D047071:beta-Glucans; C516584:micellapist; D004247:DNA