Quantitation of radiation-, chemical-, or enzyme-induced single strand breaks in nonradioactive DNA by alkaline gel electrophoresis: application to pyrimidine dimers

Journal Article · Wed Oct 01 04:00:00 EDT 1986 · Anal. Biochem.; (United States)

DOI:https://doi.org/10.1016/0003-2697(86)90599-3· OSTI ID:6941007

Freeman, S E; Blackett, A D; Monteleone, D C; Setlow, R B; Sutherland, B M; Sutherland, J C

The authors have developed an alkaline agarose gel method for quantitating single strand breaks in nanogram quantities of nonradioactive DNA. After electrophoresis together with molecular length standards, the DNA is neutralized, stained with ethidium bromide, photographed, and the density profiles recorded with a computer controller scanner. The medium lengths, number average molecular lengths, and length average molecular lengths of the DNAs can be computed by using the mobilities of the molecular length standards. The frequency of single strand breaks can then be determined by comparison of the corresponding average molecular lengths of DNAs treated and not treated with single stand break-inducing agents (radiation, chemicals, or lesion-specific endonuclease). Single stand break yields (induced at pyrimidine dimer sites in uv-irradiated human fibroblasts DNA by the dimer-specific endonuclease from Micrococcus luteus) from our method agree with values obtained for the same DNAs from alkaline sucrose gradient analysis. The method has been used to determined pyrimidine dimer yields in DNA from biopsies of human skin irradiated in situ. It will be especially useful in determining the frequency of single strand breaks (or lesions convertible to single stand breaks by specific cleaving reagents or enzymes) in small quantities of DNA from cells or tissues not amendable to radioactive labeling.

• Research Organization: Brookhaven National Lab., Upton, NY
• OSTI ID: 6941007
• Journal Information: Anal. Biochem.; (United States), Journal Name: Anal. Biochem.; (United States) Vol. 158:1; ISSN ANBCA
• Country of Publication: United States
• Language: English

Related Subjects

560120* -- Radiation Effects on Biochemicals
Cells
& Tissue Culture
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ANIMAL CELLS
BIOCHEMISTRY
CHEMISTRY
CONNECTIVE TISSUE CELLS
DNA
ELECTROMAGNETIC RADIATION
ELECTROPHORESIS
ENZYMES
ESTERASES
EXTREME ULTRAVIOLET RADIATION
FIBROBLASTS
HYDROLASES
MOLECULAR BIOLOGY
NUCLEASES
NUCLEIC ACIDS
ORGANIC COMPOUNDS
PHOSPHODIESTERASES
PYRIMIDINE DIMERS
RADIATIONS
RADIOINDUCTION
SOMATIC CELLS
STRAND BREAKS
ULTRAVIOLET RADIATION