This paper presents results on ultralong read DNA sequencing with relatively short separation times using capillary electrophoresis with replaceable polymer matrixes. In previous work, the effectiveness of mixed replaceable solutions of linear polyacrylamide (LPA) was demonstrated, and 1000 bases were routinely obtained in less than 1 h. Substantially longer read lengths have now been achieved by a combination of improved formulation of LPA mixtures, optimization of temperature and electric field, adjustment of the sequencing reaction, and refinement of the base-caller. The average molar masses of LPA used as DNA separation matrixes were measured by gel permeation chromatography and multiangle laser light scattering. Newly formulated matrixes comprising 0.5% (w/w) 270 kDa and 2% (w/w) 10 or 17 MDa LPA raised the optimum column temperature from 60 to 70 degrees C, increasing the selectivity for large DNA fragments, while maintaining high selectivity for small fragments as well. This improved resolution was further enhanced by reducing the electric field strength from 200 to 125 V/cm. In addition, because sequencing accuracy beyond 1000 bases was diminished by the low signal from G-terminated fragments when the standard reaction protocol for a commercial dye primer kit was used, the amount of these fragments was doubled. Augmenting the base-calling expert system with rules specific for low peak resolution also had a significant effect, contributing slightly less than half of the total increase in read length. With full optimization, this read length reached up to 1300 bases (average 1250) with 98.5% accuracy in 2 h for a single-stranded M13 template.

Polymers and hydrogels that swell or shrink in response to environmental stimuli such as changes in temperature, pH, or ionic strength are of interest as switchable materials for applications in biotechnology. In this paper, we show that thermoresponsive polymers offer some particular advantages as entangled matrices for DNA sequencing by capillary and microchip electrophoresis. Matrices based on conventional water-soluble polymers demand a compromise in their design for microchannel electrophoresis: whereas highly entangled solutions of high molar mass polymers provide optimal sequencing performance, their highly viscous solutions require application of high pressures to be loaded into electrophoresis microchannels. Here, we demonstrate the reproducible synthesis, precise characterization, and excellent DNA sequencing performance of high molar mass, thermoresponsive polymer matrices that exhibit a reversible, temperature-controlled "viscosity switch" from high-viscosity solutions at 25 degrees C to low-viscosity, microphase-separated colloidal dispersions at a chosen, elevated temperature. The viscosity switch decouples matrix loading and sieving properties, enabling acceleration of microchannel flow by 3 orders of magnitude. DNA sequencing separations yielding read lengths of 463 bases of contiguous sequence in 78 min with 97% base-calling accuracy can be achieved in these matrices. Switchable matrices will be particularly applicable to microfluidic devices with dynamic temperature control, which are likely to provide the next major leap in the efficiency of high-throughput DNA analysis.

We present the first optimization of linear polyacrylamide (LPA)-based DNA separation matrixes for an automated tandem microchannel single-strand conformation polymorphism (SSCP)/heteroduplex analysis (HA) method, implemented in capillary arrays dynamically coated with poly(N-hydroxyethylacrylamide) (polyDuramide). An optimized protocol for sample preparation allowed both SSCP and HA species to be produced in one step in a single tube and distinguished in a single electrophoretic analysis. A simple, two-color fluorescent sample labeling and detection strategy enabled unambiguous identification of all DNA species in the electropherogram, both single- and double-stranded. Using these protocols and a panel of 11 p53 mutant DNA samples in comparison with wild-type, we employed high-throughput capillary array electrophoresis (CAE) to carry out a systematic and simultaneous optimization of LPA weight-average molar mass (Mw) and concentration for SSCP/HA peak separation. The combination of the optimized LPA matrix (6% LPA, Mw 600 kDa) and a hydrophilic, adsorbed polyDuramide wall coating was found to be essential for resolution of CAE-SSCP/HA peaks and yielded sensitive mutation detection in all 11 p53 samples initially studied. A larger set of 32 mutant DNA specimens was then analyzed using these optimized tandem CAE-SSCP/HA protocols and materials and yielded 100% sensitivity of mutation detection, whereas each individual method yielded lower sensitivity on its own (93% for SSCP and 75% for HA). This simple, highly sensitive tandem SSCP/HA mutation detection method should be easily translatable to electrophoretic analyses on microfluidic devices, due to the ease of the capillary coating protocol and the low viscosity of the matrix.

To elucidate the impact of matrix chemical and physical properties on DNA sequencing separations by capillary electrophoresis (CE), we have synthesized, characterized and tested a controlled set of different polymer formulations for this application. Homopolymers of acrylamide and N,N-dimethylacrylamide (DMA) and copolymers of DMA and N,N-diethylacrylamide (DEA) were synthesized by free radical polymerization and purified. Polymer molar mass distributions were characterized by tandem gel permeation chromatography - laser light scattering. Polymers with different chemical compositions and similar molar mass distributions were selected and employed at the same concentration so that the variables of comparison between them were hydrophobicity and average coil size in aqueous solution. We find that the low-shear viscosities of 7% w/v polymer solutions decrease by orders of magnitude with increasing polymer hydrophobicity, while hydrophilic polymers exhibit more pronounced reductions in viscosity with increased shear. The performance of the different matrices for DNA sequencing was compared with the same sample under identical CE conditions. The longest read length was produced with linear polyacrylamide (LPA) while linear poly-N,N-dimethylacrylamide (PDMA) gave approximately 100 fewer readable bases. Read lengths with DMA/DEA copolymers were lower, and decreased with increasing DEA content. This study highlights the importance of polymer hydrophilicity for high-performance DNA sequencing matrices, through the formation of robust, highly-entangled polymer networks and the minimization of hydrophobic interactions between polymers and fluorescently-labeled DNA molecules. However, the results also show that more hydrophobic matrices offer much lower viscosities, enabling easier microchannel loading at low applied pressures.