Gad M, Machida M, Mizutani W, Ishikawa M
Joint Research Center for Atom Technology-JRCAT, Angstrom Technology Partnership, National Institute of Advanced Industrial Science and Technology (AIST), AIST Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-0046, Japan.
J Biomol Struct Dyn. 2001 Dec;19(3):471-7. doi: 10.1080/07391102.2001.10506755.
An efficient method was developed to stretch DNA molecules on an atomically flat surface for AFM imaging. This method involves anchoring DNA molecules from their 5' ends to amino silanized mica surfaces. N-Succinimidyl6-[3'-(2-pyridyldithio) propionamido]hexanoate (LC-SPDP), a heterobifunctional cross-linker with a flexible spacer arm was used for this purpose. Immobilization was carried out by introducing a thiol group to the 5' end of DNA by PCR. Thiolated molecules were then reacted with the cross linker to conjugate with its 2-pyridyl disulphide group via sulfhydryl exchange. The resulting complex was deposited on amino silanized mica where NHS-ester moiety of the cross linker reacted with the primary amino group on the surface. Samples were washed by a current of water and dried by an air jet in one direction parallel to the surface. DNA molecules were fully stretched in one direction on imaging them by AFM.
开发了一种在原子级平整表面上拉伸DNA分子以进行原子力显微镜（AFM）成像的有效方法。该方法包括将DNA分子从其5'端锚定到氨基硅烷化云母表面。N-琥珀酰亚胺基6-[3'-(2-吡啶二硫代)丙酰胺基]己酸酯（LC-SPDP），一种带有柔性间隔臂的异双功能交联剂，用于此目的。通过PCR在DNA的5'端引入巯基来进行固定。然后使硫醇化分子与交联剂反应，通过巯基交换与其2-吡啶基二硫基团共轭。将所得复合物沉积在氨基硅烷化云母上，其中交联剂的NHS-酯部分与表面上的伯氨基反应。用水流冲洗样品，并在与表面平行的一个方向上用喷气干燥。通过AFM成像时，DNA分子在一个方向上完全伸展。
