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

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Date

2003-02

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Authors

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

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East Carolina University

Abstract

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. Originally published Biophysical Journal, Vol. 84, No. 2, Feb 2003

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Biophysical Journal; 84:2 p. 1281-1290

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