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Unidirectional pulsed-field electrophoresis of single- and double-stranded DNA in agarose gels: analytical expressions relating mobility and molecular length and their application in the measurement of strand breaks |
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| Authors: | J C Sutherland D C Monteleone J H Mugavero J Trunk |
| Institution: | 1. Laboratoire de pharmacologie-toxicologie, institut de biologie en santé, CHU d’Angers, 4, rue Larrey, 49933 Angers cedex 9, France;2. Centre antipoison–toxicovigilance, CHU d’Angers, 4, rue Larrey, 49933 Angers cedex 9, France;1. Université Côte d’Azur, CNRS, INSERM, Institute of Biology Valrose (iBV), Parc Valrose, Nice, France;2. Université Côte d’Azur, CNRS, Institute Physics of Nice (INPHYNI), Ave. J. Vallot, Nice, France;1. Department of Infection Control and Prevention, Kurume University School of Medicine, 67 Asahi-machi, Kurume City, Fukuoka 830-0011, Japan;2. Division of Clinical Research, Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba City, Chiba 260-8673, Japan;1. The Pennsylvania State University, Department of Biochemistry and Molecular Biology, 107 Althouse Laboratories, University Park, PA 16802, United States;2. University of Maryland Baltimore County, 1000 Hilltop Circle, 006 Meyerhoff Chemistry Building, Baltimore, MD 21250, United States;3. Penn State Huck Institutes of the Life Sciences, 323 Life Sciences Building, University Park, PA 16802, United States;4. Perkin Elmer, 710 Bridgeport Avenue, Shelton, CT 06484, United States |
| Abstract: | Unidirectional pulsed-field electrophoresis improves the separation of single-stranded DNA molecules longer than 20 kilobases (kb) in alkaline agarose gels compared to static-field electrophoresis. The greatest improvement in separation is for molecules longer than 100 kb. The improved resolution of long molecules with unidirectional pulsed-field electrophoresis makes possible the measurement of lower frequencies of single-strand breaks. The analytical function that relates the length and mobility of single-stranded DNA electrophoresed with a static field also applies to unidirectional pulsed field separations. Thus, the computer programs used to measure single-strand breaks are applicable to both undirectional pulsed- and static-field separations. Unidirectional pulsed-field electrophoresis also improves the separation of double-stranded DNA in neutral agarose gels. The function relating molecular length and mobility for double-stranded DNA separated by unidirectional pulsed-field electrophoresis is a superset of the function for single-stranded DNA. The coefficients of this function can be determined by iterative procedures. |
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