TY - JOUR
T1 - Quantitation of radiation-, chemical-, or enzyme-induced single strand breaks in nonradioactive DNA by alkaline gel electrophoresis
T2 - Application to pyrimidine dimers
AU - Freeman, Steven E.
AU - Blackett, Anthony D.
AU - Monteleone, Denise C.
AU - Setlow, Richard B.
AU - Sutherland, Betsy M.
AU - Sutherland, John C.
N1 - Funding Information:
’ This investigation was supported by the Office of Health and Environmental Research, US Department of Energy and NIH Grants CA 26492 and CA 23096 to B.M.S. 2 Recipient of a fellowship from National Cancer Institute training Grant CA 09 12 1. 3 Present address: Molecular Neurobiology Laboratory, Department of Ophthalmic Optics, UMIST. Manchester, U.K. ’ To whom correspondence should be addressed.
PY - 1986/10
Y1 - 1986/10
N2 - We 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 controlled scanner. The median 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 strand break-inducing agents (radiation, chemicals, or lesion-specific endonuclease). Single strand 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 determine 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 strand breaks by specific cleaving reagents or enzymes) in small quantities of DNA from cells or tissues not amenable to radioactive labeling.
AB - We 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 controlled scanner. The median 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 strand break-inducing agents (radiation, chemicals, or lesion-specific endonuclease). Single strand 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 determine 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 strand breaks by specific cleaving reagents or enzymes) in small quantities of DNA from cells or tissues not amenable to radioactive labeling.
KW - DNA damage
KW - gel electrophoresis
KW - nucleic acids
KW - pyrimidine dimers
KW - single strand breaks
KW - uv
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U2 - 10.1016/0003-2697(86)90599-3
DO - 10.1016/0003-2697(86)90599-3
M3 - Article
C2 - 3799960
AN - SCOPUS:0023018781
SN - 0003-2697
VL - 158
SP - 119
EP - 129
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -