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.
Filippova
Elena M
EM
Biology Department, Brookhaven National Laboratory, Upton, New York 11973, USA.
Monteleone
Denise C
DC
Trunk
John G
JG
Sutherland
Betsy M
BM
Quake
Stephen R
SR
Sutherland
John C
JC
D017123:Bacteriophage T7 / Q000737:chemistry / Q000235:genetics; D001482:Base Composition; D001483:Base Sequence; D019457:Chromosome Breakage; D004249:DNA Damage; D004279:DNA, Viral / Q000032:analysis* / Q000737:chemistry* / Q000145:classification / Q000528:radiation effects; D005240:Feasibility Studies; D007834:Lasers; D008969:Molecular Sequence Data; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*
D004279:DNA, Viral