Investigation of ascorbate-Cu (II) induced cleavage of DNA by scanning tunneling microscopy

Scanning Tunneling Microscopy (STM) was used for the investigation of oxidative DNA damage. A PCR amplified fragment of human beta-globin gene was used as a model for time dependent cleavage reaction by ascorbate and copper. Cleavage reactions were carried out in a medium containing 0.5 microgram/20 microliters DNA, 20 nM Tris-HC1 pH, 7.8 and ascorbate-Cu (II) in the final concentrations of 1 mM and 30 microM, respectively. The mixtures were incubated at 37 degrees C for 5, 15 and 30 min. For STM studies, 3 pg/5 microliters DNA samples were deposited on the gold coated mica and dried in a water flow vacuum drier. The STM was operated in air at atmospheric pressure with a tip-to-substrate bias of 100 mV and tunneling currents of < 10 pA. Etched tips of Pt/Ir wires were used in a constant current mode. The degradated DNA structure can be distinguished from the intact DNA and the sizes of the degradation products can be identified in the STM micrographs. The size of fragments decreased from approximately 3000 A to 34 A in ascorbate-Cu (II) medium, after 30 min of incubation.