(365b) Rapid, Gel-Free Electrophoretic Separation of DNA Oligonucleotides Using Surfactant Systems: Extended Read Frame by Buffer Design

We present some significant improvements to the “micelle-ELFSE” method we have developed for the rapid electrophoretic separation of end-alkylated DNA oligonucleotides (aDNA) in the presence of nonionic surfactant micelles.  In capillary electrophoresis (CE) format, the aDNA are injected into the capillary pre-filled with a running buffer containing nonionic surfactant above its critical micelle concentration.  On application of electric field, the aDNA migrate through the capillary and attach to the micelles, retarding their mobility in a length-dependent way.  The rapid statistical sampling of micelle aggregation number ensures that a highly uniform retardation is experienced by all aDNA in the sample, yielding very sharp peaks under appropriate conditions.  As a result, DNA differing by a single base, up to 420 bases in length, can be distinguished in less than 15 minutes using commercial CE equipment.  This represents an 8-10 fold decrease in runtime compared to commercial capillary gel electrophoresis methods.

Here, we present a theory that predicts the best possible separation schemes given the timescale of micelle size fluctuations, the polydispersity of the micelles, diffusion-based broadening, injection and detection-based broadening, and wall adsorption.  Interestingly, we find that minimum run times required for each possible DNA read frame are set only by the polydispersity and fluctuation dynamics of micelles in the running buffer, not by the capillary length or electric fields used or even the size of the micelles.  This insight is crucial for the design of optimal run conditions and buffers for various bioanalysis applications.  We demonstrate that, with proper design, the micelle-ELFSE method can be implemented to separate DNA lengths well above the biased reptation limit with single-base resolution.  We present some recent applications of the micelle-ELFSE method, including identification of STR loci, miRNA profiling, and single-base-extension sequencing.