Reduced matrix viscosity in DNA sequencing by CE and microchip electrophoresis using a novel thermo‐responsive copolymer

Abstract

High viscosity is an inherent problem of sequencing matrices made of hydrophilic polymers. This problem is amplified in separations using microchips where the channels are even smaller. A novel thermal associating graft copolymer, using linear polyacrylamide (LPA) as the backbone and poly(propylene oxide) (PPO) as the graft side chain was synthesized. The injection problem could be resolved by introducing PPO side chains that can be assembled by using temperature changes but without an obvious detrimental effect on the sieving ability of the LPA. Viscosity measurement showed that these LPA-g-PPO copolymers had a transition temperature of ~ 40 °C, above which a significant increase in viscosity was observed. The sequencing performance depended on the thermal association properties of PPO and related parameters. Without optimization, a read length of 1000 bases with a single base resolution of 0.3 was achieved within an hour on an ABI 310 analyzer, using 1.8 wt% LPA-g-PPO (1.8 MDa, PPO, 0.2%). This novel thermal reversible copolymer solution can be a promising candidate as a viable matrix for DNA sequencing in capillary electrophoresis, and even more so in microchip electrophoresis.

Hydrogels in Electrophoresis: Applications and Advances

“…34,35 In addition, the modified acrylamide crosslinking matrixes were synthesized without any bubble formations. 36 To solve the dilemma of whether to choose a powerful separation hydrogel or an operation feasible polymer solution, viscosity switchable copolymer gels 37,38 have been developed. The sieving matrix could be replaced at a low viscosity state, and provide uncompromised separation performance at the high viscosity state.…”

Polymer sieving matrices in microanalytical electrophoresis

“…135 For example, thermallyresponsive (thermo-responsive) polymers have been extensively studied for DNA analysis in CGE formats because their viscosity can be tuned by temperature. 76,93,136 While concentrated highmolecular-mass polymer such as LPA can provide excellent separation efficiency, pressure as high as 1000 psi is required to ll a capillary with the polymer solution. 93 Noting that glass or plastic microuidic chips can sometimes withstand just moderate pressures (<200 psi, 136 with the exception of solventbonded PMMA 86 ), "viscosity switching" is an attractive property of a sieving matrix.…”

“…76,93,136 While concentrated highmolecular-mass polymer such as LPA can provide excellent separation efficiency, pressure as high as 1000 psi is required to ll a capillary with the polymer solution. 93 Noting that glass or plastic microuidic chips can sometimes withstand just moderate pressures (<200 psi, 136 with the exception of solventbonded PMMA 86 ), "viscosity switching" is an attractive property of a sieving matrix. Reversible viscosity switching supports gel replacement for the subsequent electrophoresis runs at the lowviscosity state, and uncompromised separation performance at the high-viscosity state (Fig.…”

“…Various polymers including HPC, 93,137 PNIPAAm [poly(N-isopropylacrylamide)], its copolymer P(NIPAAm-acrylamide-Bis acrylamide), 138 76 and graed polymer LPA-g-PPO 136 have been studied for separation applications. Among these polymers, a handful are applied to protein separation in CE formats 68,139 while many of the stimuliresponsive polymers have been actively applied for actuators, micropumps, and microvalves.…”

CGE‐laser induced fluorescence of double‐stranded DNA fragments using GelGreen dye

“…Although with lower throughput capability than the aforementioned techniques, CGE is also well suited for the simultaneous analysis of multiple DNA sequences due to its high resolving power, analysis time, and simplicity. Last developments in CGE include novel sieving matrices and additives to provide high resolution in DNA separations; novel capillary coatings ; and miniaturization into microchips . The use of LIF in CGE improves dramatically both; the LOD and linear dynamic range obtainable compared with that of UV detection .…”