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Quantifying Double-Strand Breaks and Clustered Damages in DNA by Single-Molecule Laser Fluorescence Sizing

Elena M. Filippova^*, Denise C. Monteleone^*, John G. Trunk^*, Betsy M. Sutherland^*, Stephen R. Quake and John C. Sutherland^*,

^* Biology Department, Brookhaven National Laboratory, Upton, New York 11973; Department of Applied Physics, California Institute of Technology, Pasadena, California 91125; and Department of Physics, East Carolina University, Greenville, North Carolina 27858-4353

Correspondence: Address reprint requests to Dr. John Sutherland, Dept. of Physics, East Carolina University, Greenville, NC 27858-4353. Tel.: 252-328-2023; E-mail: sutherlandj{at}mail.ecu.edu.

Fluorescence from a single DNA molecule passing through a laser beam is proportional to the size (contour length) of the molecule, and molecules of different sizes can be counted with equal efficiencies. Single-molecule fluorescence can thus determine the average length of the molecules in a sample and hence the frequency of double-strand breaks induced by various treatments. Ionizing radiation-induced frank double-strand breaks can thus be quantified by single-molecule sizing. Moreover, multiple classes of clustered damages involving damaged bases and abasic sites, alone or in combination with frank single-strand breaks, can be quantified by converting them to double-strand breaks by chemical or enzymatic treatments. For a given size range of DNA molecules, single-molecule sizing is as or more sensitive than gel electrophoresis, and requires several orders-of-magnitude less DNA to determine damage levels.

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