Wu C, Quesada M A, Schneider D K, Farinato R, Studier F W, Chu B
Department of Chemistry, State University of New York at Stony Brook 11794-3400, USA.
Electrophoresis. 1996 Jun;17(6):1103-9. doi: 10.1002/elps.1150170620.
Two preparations of linear polyacrylamide with average molecular weights of 0.37 million and 1.14 million Da, and a deuterated preparation with an average molecular weight of 1.71 million Da, were used to study the effects of molecular weight, polydispersity, and concentration on the mesh size of entangled polymers in a DNA sequencing buffer solution and their ability to resolve DNA sequencing reactions by capillary electrophoresis. The polyacrylamide concentrations were above the overlap threshold C*, the concentration above which an entangled polymer network is expected to form. Small angle neutron scattering experiments showed that between 1% and 8% polyacrylamide, the mesh size ( xi ) can be expressed by the relation xi = 2.09C-0.76, where xi is in A and C is the polymer concentration in g/mL. The mesh size depended only on the concentration and was independent of the average molecular weight of the polyacrylamide. Consistent with this result, electrophoretic mobilities of DNA moving through the polymer network depended almost entirely on the polyacrylamide concentration and not on its molecular weight or polydispersity. Although separation was little affected, band sharpness persisted to longer DNAs when the polymer network contained a higher fraction of larger polyacrylamide molecules. We postulate a dispersive effect that depends on the size of the DNA and the resiliency of the polymer network. This interpretation provides a rationale for optimizing the design of polymer solutions to sieve DNA for sequencing by capillary electrophoresis.
使用了两种平均分子量分别为37万道尔顿和114万道尔顿的线性聚丙烯酰胺制剂，以及一种平均分子量为171万道尔顿的氘代制剂，来研究分子量、多分散性和浓度对DNA测序缓冲溶液中缠结聚合物网孔大小的影响，以及它们通过毛细管电泳解析DNA测序反应的能力。聚丙烯酰胺浓度高于重叠阈值C*，即预期形成缠结聚合物网络的浓度。小角中子散射实验表明，在1%至8%的聚丙烯酰胺之间，网孔大小（ξ）可以用关系式ξ = 2.09C^(-0.76)表示，其中ξ的单位为埃，C为聚合物浓度（单位：g/mL）。网孔大小仅取决于浓度，与聚丙烯酰胺的平均分子量无关。与此结果一致，通过聚合物网络移动的DNA的电泳迁移率几乎完全取决于聚丙烯酰胺浓度，而不取决于其分子量或多分散性。尽管分离受影响较小，但当聚合物网络中含有较高比例的较大聚丙烯酰胺分子时，条带锐度对于更长的DNA持续存在。我们推测存在一种取决于DNA大小和聚合物网络弹性的分散效应。这种解释为优化聚合物溶液设计以通过毛细管电泳筛分DNA进行测序提供了理论依据。
