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首页> 外文期刊>Electrophoresis: The Official Journal of the International Electrophoresis Society >A novel thermogelling matrix for microchannel DNA sequencing based on poly-N-alkoxyalkylacrylamide copolymers.
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A novel thermogelling matrix for microchannel DNA sequencing based on poly-N-alkoxyalkylacrylamide copolymers.

机译：基于聚-N-烷氧基烷基丙烯酰胺共聚物的微通道DNA测序的新型热胶基质。

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摘要

We have developed a novel class of thermogelling polymer networks based on poly-N-alkoxyalkylacrylamides, and demonstrated their use as DNA sequencing matrices for high-throughput microchannel electrophoresis in capillary arrays. Polymers and copolymers of N-ethoxyethylacrylamide (NEEA) and N-methoxyethylacrylamide (NMEA) were synthesized by aqueous-phase free-radical polymerization and characterized by tandem gel permeation chromatography-multi-angle laser light scattering. These copolymer matrices exhibit "re-entrant"-type volume phase transitions, forming entangled networks with high shear viscosity at low (< 20 degrees C) and high (> 35 degrees C) temperatures, and undergoing a coil-to-globular transition over an intermediate temperature range (20-35 degrees C). Hence, matrix viscosity is relatively low at room temperature (25 degrees C), and increases rapidly above 35 degrees C. The material properties and phase behavior of these thermogelling polymer networks were studied by steady-shear rheometry. These matrices are easily loaded into capillary arrays at room temperature while existing as viscous fluids, but thermogel above 35 degrees C to form transparent hydrogels via a thermo-associative phase transition. The extent of the intermediate viscosity drop and the final viscosity increase depends on the composition of the copolymers. DNA sequencing by capillary array electrophoresis with four-color laser-induced fluorescence (LIF) detection shows that these thermogelling networks provide enhanced resolution of both small and large DNA sequencing fragments and longer sequencing read lengths, in comparison to appropriate control (closely related, nonthermogelling) polymer networks. In particular, a copolymer comprised of 90% w/w NMEA and 10% w/w NEEA, with a molecular mass of approximately 2 MDa, delivers around 600 bases at 98.5% base-calling accuracy in 100 min of electrophoresis.
机译：我们已经开发出了一类基于聚N-烷氧基烷基丙烯酰胺的新型热胶凝聚合物网络，并证明了它们可作为毛细管阵列中高通量微通道电泳的DNA测序基质。通过水相自由基聚合合成了N-乙氧基乙基丙烯酰胺（NEEA）和N-甲氧基乙基丙烯酰胺（NMEA）的聚合物和共聚物，并通过串联凝胶渗透色谱-多角度激光散射进行了表征。这些共聚物基质表现出“凹角”型体积相变，在低（<20摄氏度）和高（> 35摄氏度）温度下形成具有高剪切粘度的缠结网络，并在整个过程中经历了盘绕至球形转变中间温度范围（20-35摄氏度）。因此，基质粘度在室温（25摄氏度）下相对较低，并在35摄氏度以上迅速增加。这些热胶凝聚合物网络的材料特性和相行为通过稳定剪切流变法进行了研究。这些基质很容易在室温下以粘性流体的形式加载到毛细管阵列中，但是在35摄氏度以上的热凝胶通过热缔合相转变形成透明的水凝胶。中间粘度下降和最终粘度增加的程度取决于共聚物的组成。通过四色激光诱导的荧光（LIF）检测通过毛细管阵列电泳进行的DNA测序表明，与适当的对照相比（与之密切相关的非热凝胶），这些热凝胶网络可提供大小不一的DNA测序片段的增强分辨率和更长的测序读取长度）聚合物网络。特别地，由90％w / w NMEA和10％w / w NEEA组成的共聚物，其分子量约为2 MDa，在100分钟的电泳中以98.5％的碱基检出准确度传递了约600个碱基。

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