Belozerova Irina, Ge Dongbiao, Levicky Rastislav
Polytechnic Institute of New York University, New York, NY, USA.
Methods Mol Biol. 2013;1025:127-36. doi: 10.1007/978-1-62703-462-3_10.
Thermal denaturation, or melting, measurements are a classic technique for analysis of thermodynamics of nucleic base driven associations in solution, as well as of interactions between nucleic acids and small molecule ligands such as drugs or carcinogens. Performed on surface-immobilized DNA films, this well-established technique can help understand how energetics of surface hybridization relate to those in solution, as well as provide high-throughput platforms for screening of small molecule ligands. Here we describe methods for measuring DNA melting transitions at solid/liquid interfaces with focus on the role of immobilization chemistry, including a common "immobilization-through-self-assembly" approach that is effective at moderate temperatures, and a thermo-stable approach based on polymer-supported DNA monolayers that can be used at elevated temperatures. We also discuss conditions necessary for reversible measurements, as signified by superimposition of the association (cooling) and dissociation (heating) transitions of immobilized DNA strands.
热变性或解链测量是一种经典技术，用于分析溶液中核酸碱基驱动的缔合热力学，以及核酸与小分子配体（如药物或致癌物）之间的相互作用。在表面固定的DNA薄膜上进行这种成熟的技术，有助于理解表面杂交的能量学与溶液中的能量学之间的关系，还能提供用于筛选小分子配体的高通量平台。在此，我们描述了在固/液界面测量DNA解链转变的方法，重点关注固定化学的作用，包括一种在中等温度下有效的常见“通过自组装固定”方法，以及一种基于聚合物支撑的DNA单分子层的热稳定方法，该方法可在高温下使用。我们还讨论了可逆测量所需的条件，这由固定化DNA链的缔合（冷却）和解离（加热）转变的叠加表示。
