Jetha Nahid N, Wiggin Matthew, Marziali Andre
Department of Physics and Astronomy, University of British Columbia, V6T 1Z1, Vancouver, BC, Canada.
Methods Mol Biol. 2009;544:129-50. doi: 10.1007/978-1-59745-483-4_10.
Force spectroscopy can be applied using nanopores to study charged molecules such as nucleic acids. This technique can be used to study the binding energy of a DNA duplex by threading an anchored single-stranded DNA (ssDNA) probe molecule through a nanopore (having a diameter large enough to accommodate only a single strand) and allowing target DNA on the backside of the pore to hybridize to the probe. Electric potential can be used to apply a force to the charged ssDNA in a direction tending to translocate the duplex through the pore. If the pore is only large enough to accept ssDNA, the duplex must dissociate for the probe to escape the pore. The dissociation time of the duplex can therefore be measured under applied force, and (provided that enough dissociation events have been recorded) a characteristic time scale for dissociation can be determined. In this chapter, we present a detailed protocol for performing nanopore force spectroscopy on DNA duplexes using one or more alpha-hemolysin nanopores. We present the details of the measurement of the duplex survival probability under force, and show that dissociation time scales for duplexes that are perfectly complimentary differ by greater than approximately two orders of magnitude from those containing a single sequence mismatch, offering opportunities for sequence detection.
力谱技术可通过纳米孔来应用，以研究诸如核酸等带电分子。该技术可用于研究DNA双链体的结合能，方法是将一个锚定的单链DNA（ssDNA）探针分子穿过一个纳米孔（其直径大到仅能容纳单链），并使孔背面的目标DNA与探针杂交。电势可用于沿倾向于使双链体穿过孔的方向对带电的ssDNA施加力。如果孔仅大到足以容纳ssDNA，双链体必须解离，探针才能从孔中逸出。因此，可以在施加力的情况下测量双链体的解离时间，并且（前提是记录了足够多的解离事件）可以确定解离的特征时间尺度。在本章中，我们给出了使用一个或多个α-溶血素纳米孔对DNA双链体进行纳米孔力谱分析的详细方案。我们给出了在力作用下双链体存活概率测量的细节，并表明完全互补的双链体的解离时间尺度与包含单个序列错配的双链体的解离时间尺度相差大于约两个数量级，这为序列检测提供了机会。
