(327c) DNA Sequencing and Separation in Free Solution Using Engineered Drag-Tags

End-Labeled Free-Solution Electrophoresis (ELFSE) is a strategy for size-based separation of DNA in free solution that promises improved performance while avoiding the fundamental and practical limitations associated with electrophoresis in a gel or sieving matrix. In ELFSE, each DNA molecule in a mixture is conjugated to a polymeric "drag-tag" that modifies the free-draining properties of the DNA, enabling electrophoretic separation without a polymer matrix. Very rapid separations with long read lengths are theoretically possible using ELFSE. Since no viscous sieving matrix is necessary, ELFSE should simplify the transition of DNA sequencing separations onto integrated microfluidic devices currently under development.

The primary obstacle to competitive sequencing with ELFSE has historically been the availability of drag-tags that are sufficiently large and monodisperse for high-resolution separation of a wide range of sizes of DNA. We present here key experimental advances in the implementation of ELFSE. We have developed and tested new drag-tags, including monodisperse synthetic polymers, as well as repetitive polypeptides or "protein polymers" engineered specifically for ELFSE. We have used these engineered drag-tags for DNA sequencing, and although the read lengths are not yet comparable to matrix-based sequencing, the drag-tags we have created are a significant improvement over previous generations of drag-tags, and represent an important step along the way to truly long-read sequencing.

In addition to DNA sequencing, we report several other novel uses for ELFSE, including a highly multiplexed genotyping assay based on a single-base extension reaction that allows rapid, high-throughput analysis of single nucleotide polymorphisms or point mutations. We have also performed ELFSE separations with polymeric drag-tags at both ends of the DNA chain, an approach that offers enhanced hydrodynamic drag and extends the range of separations possible with ELFSE. ELFSE sequencing and genotyping are particularly exciting on microfluidic devices, where short separation distances and high electric fields enable very rapid separations. We are actively developing methodologies for microfluidic ELFSE separations, with ongoing optimization of sample preconcentration and injection, as well as microchannel wall coating and chip architecture .