Method for orienting DNA molecules on mica surfaces in one direction for atomic force microscopy imaging.
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.
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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. gmuhamme@jrcat.or.jp
Machida
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Mizutani
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Ishikawa
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D000538:Aluminum Silicates / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000648:ultrastructure*; D018625:Microscopy, Atomic Force / Q000379:methods*; D009690:Nucleic Acid Conformation; D012821:Silanes / Q000737:chemistry
D000538:Aluminum Silicates; D003432:Cross-Linking Reagents; D012821:Silanes; D004247:DNA; C011934:mica