Ultrafast DNA sequencing on a microchip by a hybrid separation mechanism that gives 600 bases in 6.5 minutes.

To realize the immense potential of large-scale genomic sequencing after the completion of the second human genome (Venter's), the costs for the complete sequencing of additional genomes must be dramatically reduced. Among the technologies being developed to reduce sequencing costs, microchip electrophoresis is the only new technology ready to produce the long reads most suitable for the de novo sequencing and assembly of large and complex genomes. Compared with the current paradigm of capillary electrophoresis, microchip systems promise to reduce sequencing costs dramatically by increasing throughput, reducing reagent consumption, and integrating the many steps of the sequencing pipeline onto a single platform. Although capillary-based systems require approximately 70 min to deliver approximately 650 bases of contiguous sequence, we report sequencing up to 600 bases in just 6.5 min by microchip electrophoresis with a unique polymer matrix/adsorbed polymer wall coating combination. This represents a two-thirds reduction in sequencing time over any previously published chip sequencing result, with comparable read length and sequence quality. We hypothesize that these ultrafast long reads on chips can be achieved because the combined polymer system engenders a recently discovered "hybrid" mechanism of DNA electromigration, in which DNA molecules alternate rapidly between repeating through the intact polymer network and disrupting network entanglements to drag polymers through the solution, similar to dsDNA dynamics we observe in single-molecule DNA imaging studies. Most importantly, these results reveal the surprisingly powerful ability of microchip electrophoresis to provide ultrafast Sanger sequencing, which will translate to increased system throughput and reduced costs.

DNA migration mechanism analyses for applications in capillary and microchip electrophoresis.

In 2009, electrophoretically driven DNA separations in slab gels and capillaries have the sepia tones of an old-fashioned technology in the eyes of many, even while they remain ubiquitously used, fill a unique niche, and arguably have yet to reach their full potential. For comic relief, what is old becomes new again: agarose slab gel separations are used to prepare DNA samples for "next-gen" sequencing platforms (e.g. the Illumina and 454 machines) - dsDNA molecules within a certain size range are "cut out" of a gel and recovered for subsequent "massively parallel" pyrosequencing. In this review, we give a Barron lab perspective on how our comprehension of DNA migration mechanisms in electrophoresis has evolved, since the first reports of DNA separations by CE ( approximately 1989) until now, 20 years later. Fused-silica capillaries and borosilicate glass and plastic microchips quietly offer increasing capacities for fast (and even "ultra-fast"), efficient DNA separations. While the channel-by-channel scaling of both old and new electrophoresis platforms provides key flexibility, it requires each unique DNA sample to be prepared in its own micro or nanovolume. This Achilles' heel of electrophoresis technologies left an opening through which pooled sample, next-gen DNA sequencing technologies rushed. We shall see, over time, whether sharpening understanding of transitions in DNA migration modes in crosslinked gels, nanogel solutions, and uncrosslinked polymer solutions will allow electrophoretic DNA analysis technologies to flower again. Microchannel electrophoresis, after a quiet period of metamorphosis, may emerge sleeker and more powerful, to claim its own important niche applications.

The incorporation of extracellular matrix proteins in protein polymer hydrogels to improve encapsulated beta-cell function.

Biomaterial encapsulation of islets has been proposed to improve the long-term success of islet transplantation by recreating a suitable microenvironment and enhancing cell-matrix interactions that affect cellular function. Protein polymer hydrogels previously showed promise as a biocompatible scaffold by maintaining high cell viability. Here, enzymatically-crosslinked protein polymers were used to investigate the effects of varying scaffold properties and of introducing ECM proteins on the viability and function of encapsulated MIN6 ß-cells. Chemical and mechanical properties of the hydrogel were modified by altering the protein concentrations while collagen IV, fibronectin, and laminin were incorporated to reestablish cell-matrix interactions lost during cell isolation. Rheology indicated all hydrogels formed quickly, resulting in robust, elastic hydrogels with Young's moduli similar to soft tissue. All hydrogels tested supported both high MIN6 ß-cell viability and function and have the potential to serve as an encapsulation platform for islet cell delivery in vivo.

Modular enzymatically crosslinked protein polymer hydrogels for in situ gelation.

Biomaterials that mimic the extracellular matrix in both modularity and crosslinking chemistry have the potential to recapitulate the instructive signals that ultimately control cell fate. Toward this goal, modular protein polymer-based hydrogels were created through genetic engineering and enzymatic crosslinking. Animal derived tissue transglutaminase (tTG) and recombinant human transglutaminase (hTG) enzymes were used for coupling two classes of protein polymers containing either lysine or glutamine, which have the recognition substrates for enzymatic crosslinking evenly spaced along the protein backbone. Utilizing tTG under physiological conditions, complete crosslinking occurred within 2 min, as determined by particle tracking microrheology. Hydrogel composition impacted the elastic storage modulus of the gel over 4-fold and also influenced microstructure and degree of swelling, but did not appreciably effect degradation by plasmin. Mouse 3T3 and primary human fibroblasts were cultured in both 2- and 3-dimensions without a decrease in cell viability and displayed spreading in 2D. The properties, which are controlled through the specific nature of the protein polymer precursors, render these gels valuable for in situ therapies. Furthermore, the modular hydrogel composition allows tailoring of mechanical and physical properties for specific tissue engineering applications.

Hydrophobically modified polyacrylamide block copolymers for fast, high-resolution DNA sequencing in microfluidic chips.

By using a microfluidic electrophoresis platform to perform DNA sequencing, genomic information can be obtained more quickly and affordably than the currently employed capillary array electrophoresis instruments. Previous research in our group has shown that physically cross-linked, hydrophobically modified polyacrylamide matrices separate dsDNA more effectively than linear polyacrylamide (LPA) solutions. Expanding upon this work, we have synthesized a series of LPA-co-dihexylacrylamide block copolymers specifically designed to electrophoretically sequence ssDNA quickly and efficiently on a microfluidic device. By incorporating very small amounts of N,N-dihexylacrylamide, a hydrophobic monomer, these copolymer solutions achieved up to approximately 10% increases in average DNA sequencing read length over LPA homopolymer solutions of matched molar mass. Additionally, the inclusion of the small amount of hydrophobe does not significantly increase the polymer solution viscosities, relative to LPA solutions, so that channel loading times between the copolymers and the homopolymers are similar. The resulting polymer solutions are capable of providing enhanced sequencing separations in a short period of time without compromising the ability to rapidly load and unload the matrix from a microfluidic device.

Stochastic single-molecule videomicroscopy methods to measure electrophoretic DNA migration modalities in polymer solutions above and below entanglement.

We have studied the effects of polymer molar mass and concentration on the electrophoretic migration modalities of individual molecules of DNA in LPA, HEC, and PEO solutions via epifluorescent videomicroscopy. While both transient entanglement coupling (TEC) and reptation have been studied in the past, the transition between them has not. Understanding this transition will allow for polymer network properties to be optimized to enhance the speed and resolution of DNA separations in microfluidic devices. Near the overlap threshold concentration, C*, TEC is the dominant observed mode of DNA migration, and the observation frequency of TEC increases with increasing polymer molar mass. As polymer concentration is increased, observed TEC events reduce to zero while DNA reptation events become the only detected mechanism. Individual DNA molecules undergoing both migration mechanisms were counted in solutions of varying polymer molar masses and concentrations and were plotted against a dimensionless polymer concentration, C/C*. The data for LPA reduce to form universal curves with a sharp increase in DNA reptation at approximately 6.5C*. Analogous transition concentrations for PEO and HEC were observed at 5C* and 3.5C*, respectively, reflecting the different physical properties of these polymers. This transition correlates closely with the polymer network entanglement concentration, Ce, as measured by rheological techniques. The electrophoretic mobility of lambda-DNA in LPA polymer solutions was also measured and shows how a balance can be struck between DNA resolution and separation speed by choosing the desired prevalence of DNA reptation.