(327a) How Is It Possible to Sequence 600 Bases of DNA in 6.5 Minutes? The Central Role of Carefully Engineered Polymer Networks and Coatings in Microchip Electrophoresis

Electrophoretic DNA sequencing remains the only technology that can deliver the long reads that are necessary for the sequencing of complex mammalian and plant genomes. We report on our work to create enabling technologies and materials for microfluidic chip electrophoresis sequencers, which promise to replace capillary array electrophoresis for high-throughput DNA sequencing. Low-viscosity DNA sequencing matrices are advantageous for microchannel electrophoresis systems since these polymer solutions can be easily pumped into the channels at relatively low applied pressures in only a few minutes. Dynamic (adsorptive) polymer coatings are also ideal for microfluidic platforms; they are much easier to apply and give virtually 100% yield of functional coatings, and are substantially more stable than covalently linked coatings. We have previously demonstrated sequencing reads of over 500 bases in 5-6 minutes using a poly(N,N-dimethylacrylamide) (pDMA) polymer matrix in short, 7.5-cm chip channels dynamically coated with poly(N-hydroxyethyl-acrylamide) (pHEA). These sequencing times are much shorter than any previous chip sequencing result (the previous speed record for > 500 base reads was 15-18 minutes). Through further development of polymer and matrix properties, we have now achieved four-color sequencing reads up to 600 bases in only 6.5 minutes (with 98.5% accuracy of base-calling), which is the longest sequencing read ever obtained in such a short separation distance. We show that this surprisingly fast sequencing is obtained by a hybrid separation mechanism, which combines both DNA reptation and transient entanglement coupling, explaining the faster and better sequencing performance of our pDMA networks relative to commercially available matrices. We discuss the DNA and polymer dynamics responsible for the excellent performance of these pDMA matrices and discuss the results in terms of the separation mechanism of DNA in different types of polymer solutions we have studied, making direct comparisons with what was previously the gold standard matrix for chip-based sequencing, linear polyacrylamide. Insights gained from these studies are being used to further optimize matrix properties and separation conditions for the further development of microchip-based DNA sequencing systems.