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

Abstract

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 notmore » amendable to radioactive labeling.« less

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Lab., Upton, NY
OSTI Identifier:
6941007
Resource Type:
Journal Article
Journal Name:
Anal. Biochem.; (United States)
Additional Journal Information:
Journal Volume: 158:1
Country of Publication:
United States
Language:
English
Subject:
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.; DNA; ELECTROPHORESIS; NUCLEASES; BIOCHEMISTRY; PYRIMIDINE DIMERS; MOLECULAR BIOLOGY; RADIOINDUCTION; EXTREME ULTRAVIOLET RADIATION; FIBROBLASTS; STRAND BREAKS; ANIMAL CELLS; CHEMISTRY; CONNECTIVE TISSUE CELLS; ELECTROMAGNETIC RADIATION; ENZYMES; ESTERASES; HYDROLASES; NUCLEIC ACIDS; ORGANIC COMPOUNDS; PHOSPHODIESTERASES; RADIATIONS; SOMATIC CELLS; ULTRAVIOLET RADIATION; 560120* - Radiation Effects on Biochemicals, Cells, & Tissue Culture

Citation Formats

Freeman, S E, Blackett, A D, Monteleone, D C, Setlow, R B, Sutherland, B M, and Sutherland, J C. Quantitation of radiation-, chemical-, or enzyme-induced single strand breaks in nonradioactive DNA by alkaline gel electrophoresis: application to pyrimidine dimers. United States: N. p., 1986. Web. doi:10.1016/0003-2697(86)90599-3.
Freeman, S E, Blackett, A D, Monteleone, D C, Setlow, R B, Sutherland, B M, & Sutherland, J C. Quantitation of radiation-, chemical-, or enzyme-induced single strand breaks in nonradioactive DNA by alkaline gel electrophoresis: application to pyrimidine dimers. United States. https://doi.org/10.1016/0003-2697(86)90599-3
Freeman, S E, Blackett, A D, Monteleone, D C, Setlow, R B, Sutherland, B M, and Sutherland, J C. 1986. "Quantitation of radiation-, chemical-, or enzyme-induced single strand breaks in nonradioactive DNA by alkaline gel electrophoresis: application to pyrimidine dimers". United States. https://doi.org/10.1016/0003-2697(86)90599-3.
@article{osti_6941007,
title = {Quantitation of radiation-, chemical-, or enzyme-induced single strand breaks in nonradioactive DNA by alkaline gel electrophoresis: application to pyrimidine dimers},
author = {Freeman, S E and Blackett, A D and Monteleone, D C and Setlow, R B and Sutherland, B M and Sutherland, J C},
abstractNote = {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.},
doi = {10.1016/0003-2697(86)90599-3},
url = {https://www.osti.gov/biblio/6941007}, journal = {Anal. Biochem.; (United States)},
number = ,
volume = 158:1,
place = {United States},
year = {Wed Oct 01 00:00:00 EDT 1986},
month = {Wed Oct 01 00:00:00 EDT 1986}
}