A novel thermogelling matrix for microchannel DNA sequencing based on poly-N-alkoxyalkylacrylamide copolymers
A novel thermogelling matrix for microchannel DNA sequencing based on poly-N-alkoxyalkylacrylamide copolymers
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DOI：
10.1002/elps.200305670
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发表时间：
2003-12-01
期刊：
影响因子：
2.9
通讯作者：
Barron, AE
中科院分区：
文献类型：
--
作者：
Kan, CW;Doherty, EAS;Barron, AE
我们已经开发了一类新的热凝胶聚合物网络的基础上聚，N-烷氧基烷基丙烯酰胺，并证明了它们的使用作为DNA测序矩阵的高通量微通道电泳在毛细管阵列。采用水相自由基聚合法合成了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，并用串联凝胶渗透色谱-多角度激光光散射对其进行了表征。这些共聚物基质表现出“重入”型体积相变，在低温（<20 ℃）和高温（> 35 ℃）下形成具有高剪切粘度的缠结网络，并在中间温度范围（20- 35 ℃）内经历“线圈到球状”、低临界溶解温度（LCST）样相变。因此，基质粘度在室温（25 ℃）下相对较低，并且在35 ℃以上迅速增加。用稳态剪切流变仪研究了这些热凝胶聚合物网络的材料性质和相行为。这些基质在室温下很容易加载到毛细管阵列中，同时作为粘性流体存在，但在35 ℃以上热凝胶化，通过热缔合相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。通过毛细管阵列电泳与四色激光诱导荧光（LIF）检测的DNA测序表明，这些热凝胶网络提供了增强的分辨率的小和大的DNA测序片段和更长的测序读长，在适当的控制（密切相关，nonthermogelating）聚合物网络相比。特别地，由90%w/w NMEA和10%w/w NEEA组成的共聚物，具有类似于2 MDa的分子量，在100分钟的电泳中以98.5%的碱基识别准确度递送约600个碱基。
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 ( < 20degreesC) and high ( > 35degreesC) temperatures, and undergoing a "coil-to-globular", lower critical solution temperature (LCST)-like phase transition over an intermediate temperature range (20-35degreesC). Hence, matrix viscosity is relatively low at room temperature (25degreesC), and increases rapidly above 35degreesC. 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 35degreesC 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 similar to 2 MDa, delivers around 600 bases at 98.5% base-calling accuracy in 100 min of electrophoresis.