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

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 200