Polymer Matrices and Dynamic Coatings for Ultra-fast DNA Sequencing Separations by Electrophoresis in Glass Microfluidic ChipsPublic Deposited
Separation of DNA molecules by electrophoresis through entangled polymer solutions continues to be an important tool for genetic analysis, especially Sanger-based DNA sequencing. Separations of DNA on microfluidic devices are much more efficient than in capillary electrophoresis (CE) systems, such that the implementation of DNA sequencing onto microchips promises to reduce both time and costs for these assays. The general focus of this field has been to develop the hardware for these instruments while assuming that the commercially available polymer materials for separating DNA fragments and passivating the channel walls will translate directly from CE instruments to microchips, and new materials for separation have not been a priority for these groups. However, many published instrumentation studies fail to reach their potential due to poor performance of separation matrices and wall coatings. The aim of this research is to develop polymer systems for the high quality sequencing of DNA in microfluidic chip devices by electrophoresis. The combination of a polymer matrix based on poly(N,N-dimethylacrylamide) (pDMA) and a microchannel wall coating consisting of adsorbed chains of the poly(N-hydroxyethylacrylamide) (pHEA) are described, which enables very fast sequencing of DNA in glass microfluidic chips. The high performance of this system is discussed in terms of the underlying mechanisms of DNA migration in polymer matrices including the hypothesis for a new separation mechanism in this polymer system that allows for fast, high quality DNA sequencing separations. The results with these materials are compared to results from commercially available matrices and coatings. Furthermore, it is shown that the chemical and physical properties of the polymer matrices have a significant influence on peak widths, and thereby resolution and read lengths, of eluting DNA sequencing fragments. The need for new sequencing matrices developed specifically for microfluidic sequencing systems is demonstrated by showing that peak broadening by dispersion limits resolution in microchips, a factor that is much less critical in capillary-based separations. Finally, this polymer system is used to demonstrate the sequencing of HIV samples isolated from human blood samples for the determination of the resistance or susceptibility of the virus to FDA approved anti-retroviral therapeutics.