IMPROVED SEPARATION MEDIA FOR ELECTROPHORESIS

改进的电泳分离介质

• 批准号: 2519138
• 负责人: Benjamin Chu
• 金额: $ 13.18万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-30 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2519138
• 关键词: DNA; analytical method; birefringences; copolymer I; electrophoresis; gel electrophoresis; light scattering; low angle X ray diffraction analysis; method development; micelles; nonblood rheology; nuclear magnetic resonance spectroscopy; nucleic acid sequence; polymerization; solutions; viscosity

In DNA sequencing analysis by means of electrophoresis, the separation medium could use further improvements. The long-term objective of the proposed project is to develop a better separation medium which can overcome many of the problems related to the present-day polyacrylamide solutions or gels: polymer solutions are often too viscous to be filled in capillary tubings; the polymerization in situ approach is not so reproducible; shelf-life for polymer gels is limited. The central theme is to take advantage of the self-assembly behavior of block copolymers so that predesigned polymer networks can be formulated by considering the chemical nature of the blocks, the total chain length, the block length ratio and the molecular architecture of the blocks in a selective solvent which is the buffer solution used in DNA sequencing analysis. In the self-assembled polymer networks, it is expected that several desirable properties can be designed into the properties of the separation medium. For examples, (1) at temperatures outside the operating temperature range for electrophoresis, the low molecular weight block copolymer (e.g. Pluronic polyols) solution has a low viscosity for ease of filling in capillary tubings or thin slabs, and (2) at the operating temperatures for electrophoresis, the self-assembled supramolecules can form gels of predetermined structures suitable for DNA sequencing analysis. In order to provide a basis for the design of a better separation medium, the structures of these entangled polymer chains (or gels) will be studied by means of physical techniques, such as viscosimetry, rheometry, laser light scattering, small angle neutron scattering, small angle x-ray scattering, C13 and proton NMR, and be correlated with electrophoresis results. By using the same physical techniques, it is proposed to study the effects of polydispersity on polymer chain entanglements and the use of a mixed polymer (e.g. polyacrylamide) solution/agarose gel approach which will be used to examine whether larger mesh size polymer networks can be achieved in order to resolve larger size DNA fragments. The agarose gel is used to restrict the translational motions of polyacrylamide chains so as to control the viscosity of the separation medium and the polymer chain dynamics.

在通过电泳进行的 DNA 测序分析中，分离 媒体可以使用进一步的改进。 该组织的长期目标 拟议的项目是开发一种更好的分离介质，可以 克服了与当今聚丙烯酰胺相关的许多问题 溶液或凝胶：聚合物溶液通常太粘稠而无法填充 毛细管；原位聚合方法则不然 可重现；聚合物凝胶的保质期是有限的。中心主题是 利用嵌段共聚物的自组装行为 预先设计的聚合物网络可以通过考虑来制定 嵌段的化学性质、总链长度、嵌段长度 选择性溶剂中嵌段的比例和分子结构 这是DNA测序分析中使用的缓冲溶液。 在 自组装聚合物网络，预计有几种理想的 特性可以被设计成分离介质的特性。 例如，(1) 在工作温度范围之外的温度下 对于电泳，低分子量嵌段共聚物（例如 Pluronic 多元醇）溶液粘度低，易于填充 毛细管或薄板，以及 (2) 在工作温度下 电泳时，自组装超分子可形成凝胶 适用于 DNA 测序分析的预定结构。 为了给更好的分离介质的设计提供依据， 将研究这些缠结的聚合物链（或凝胶）的结构 通过物理技术，例如粘度测定法、流变测定法、激光 光散射、小角中子散射、小角X射线 散射、C13 和质子 NMR，并与电泳相关 结果。 通过使用相同的物理技术，建议研究其影响 聚合物链缠结的多分散性及其使用 聚合物（例如聚丙烯酰胺）溶液/琼脂糖凝胶方法 用于检查是否可以实现更大网目尺寸的聚合物网络 以解析更大尺寸的DNA片段。琼脂糖凝胶用于 限制聚丙烯酰胺链的平移运动，从而 控制分离介质和聚合物链的粘度 动力学。