TY - JOUR
T1 - Quantitation of supercoiled DNA cleavage in nonradioactive DNA
T2 - Application to lonizing radiation and synthetic endonuclease cleavage
AU - Sutherland, Betsy M.
AU - Bennett, Paula V.
AU - Conlon, Kim
AU - Epling, Gary A.
AU - Sutherland, John C.
N1 - Funding Information:
1 Research supported by a grant from the Human Genome Project of the U.S. Department of Energy to B.M.S. and G.A.E., NIH Grants CA 23096 to B.M.S. and HG00371 to J.C.S., American Cancer Society Institutional Grant IRG-IN152C to G.A.E., and the Office of Health and Environmental Research of the U.S. Department of Energy. The U.S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, governmental purposes, is acknowledged.
PY - 1992/2/14
Y1 - 1992/2/14
N2 - Quantitation of the conversion of nonradioactive supercoiled DNA to its open circular or linear forms on ethidium-stained electrophoretic gels has been difficult because of differential binding of ethidium to supercoiled DNA vs other forms under different conditions and the nonlinear response of photographic film. We have developed methods for adding a linear DNA as an internal fluorescence standard to "normalize" the quantity of DNA loaded into each lane of a gel. Inclusion of a linear normalizing DNA in samples before partitioning for individual supercoil cleavage reactions allows the quantitation of the resultant species, is technically easy, and does not require quantitative application of the sample to the gel. If the presence of a normalizing DNA during supercoil cleavage is undesirable, the addition of a normalizing plasmid to each sample after supercoil cleavage (but before electrophoresis) or the quantitative application of samples containing test DNA alone to the gel gives similar data, but with increased variability. We use the normalizing DNA method in cleavage by a physical agent (ionizing radiation) and in a more complex situation, by a protein-based, light-dependent synthetic endonuclease. We show how the fraction of intact supercoiled DNA can be calculated from measurement of the cleaved and normalizing species only. The method also can be used in reactions involving the depletion of one DNA species, whether supercoiled or not, such as protein-DNA interactions as detected by gel retardation assays.
AB - Quantitation of the conversion of nonradioactive supercoiled DNA to its open circular or linear forms on ethidium-stained electrophoretic gels has been difficult because of differential binding of ethidium to supercoiled DNA vs other forms under different conditions and the nonlinear response of photographic film. We have developed methods for adding a linear DNA as an internal fluorescence standard to "normalize" the quantity of DNA loaded into each lane of a gel. Inclusion of a linear normalizing DNA in samples before partitioning for individual supercoil cleavage reactions allows the quantitation of the resultant species, is technically easy, and does not require quantitative application of the sample to the gel. If the presence of a normalizing DNA during supercoil cleavage is undesirable, the addition of a normalizing plasmid to each sample after supercoil cleavage (but before electrophoresis) or the quantitative application of samples containing test DNA alone to the gel gives similar data, but with increased variability. We use the normalizing DNA method in cleavage by a physical agent (ionizing radiation) and in a more complex situation, by a protein-based, light-dependent synthetic endonuclease. We show how the fraction of intact supercoiled DNA can be calculated from measurement of the cleaved and normalizing species only. The method also can be used in reactions involving the depletion of one DNA species, whether supercoiled or not, such as protein-DNA interactions as detected by gel retardation assays.
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U2 - 10.1016/0003-2697(92)90176-8
DO - 10.1016/0003-2697(92)90176-8
M3 - Article
C2 - 1320349
AN - SCOPUS:0026565945
SN - 0003-2697
VL - 201
SP - 80
EP - 86
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -