Fluorometric determination of DNA in agarose gels: usefulness for measurement of double-strand breaks in nonlabeled cells by pulsed-field gel electrophoresis.
Quantitative measurement of DNA in agarose gels, particularly as needed for measurement of double-strand breaks induced by agents such as radiation, usually involves the use of radioactively labeled DNA. Thus its usefulness is usually limited to growing cells which incorporate radiolabeled thymidine into DNA. To circumvent this problem, we have developed a fluorometric technique for quantitative estimation of DNA in the presence of large amounts of agarose. Gel slices are solubilized with concentrated sodium perchlorate and DNA is selectively precipitated with cadmium chloride. The amount of DNA can then be estimated with 3,5-diaminobenzoic acid. Determination of DNA is linear in the range 10 ng to 1 microgram or more. We describe the application of this technique to the measurement of 60Co gamma-ray-induced double-strand breaks by pulsed-field gel electrophoresis. Our results are essentially identical to those obtained using radiolabeled DNA.
Ottawa Regional Cancer Centre, University of Ottawa, Ontario, Canada.
J K
JK
Sandhu
H C
HC
Birnboim
D062365:Aminobenzoates; D000818:Animals; D016466:CHO Cells; D002104:Cadmium; D006224:Cricetinae; D004247:DNA / Q000302:isolation & purification* / Q000528:radiation effects; D004249:DNA Damage; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods*; D006098:Granulocytes; D006801:Humans
D062365:Aminobenzoates; D002104:Cadmium; C017856:3,5-diaminobenzoic acid; D004247:DNA