Identification of PCR Products Using PNA Amphiphiles in Micellar Electrokinetic Chromatography

Abstract

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.

Application of nucleic acid–lipid conjugates for the programmable organisation of liposomal modules

“…These PNA amphiphiles bind transiently with micelles and vesicles, particularly when bound to a complementary DNA sequence. This allows micelles to be PNA n electrokinetic chromatography, allowing the rapid separation of unlabelled DNA based upon their length and sequence [48,[50][51][52].…”

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

“…All MEKC datasets were fit according to the model proposed by Grosser et al [7]. In this treatment, the effective mobility of the PNAA-tagged DNA in the presence of micelles (m eff ), is simply a weighted average of the intrinsic mobility of the bound target, m …”

“…This quantity is not the observed electrophoretic mobility, but rather a limiting case achieved as the fraction of time the DNA spends attached to a micelle approaches unity. That fraction (fmic) increases with the concentration of micelles in the running buffer ([M]) [7]:…”

“…Recently, we proposed the use of surfactant micelles as ELFSE drag-tags [7]. Micelles are water-soluble and form fairly monodisperse populations of structures with a tunable size and morphology.…”

“…We have recently demonstrated the use of nonionic micelles of TX-100 as ELFSE drag-tags in CE [7]. To maintain the interaction of micelles with alkylated DNA, it is necessary to include micelles in the running buffer in a manner similar to MEKC methods [15,16].…”