Use of the diaminobenzoic acid fluorescence assay in conjunction with uv absorbance as a means of quantifying and ascertaining the purity of a DNA preparation.
In the course of a study conducted to determine the correlation between covalently bound DNA-ethyl adducts and specific locus mutation induction in maize (W. E. Schy and M. J. Plewa Mutat. Res., 211, 231-241), it was necessary to accurately quantify and ascertain the purity of small amounts of DNA isolated from germinating maize kernels. DNA was purified from leaf primordial tissue that was dissected from germinating maize kernels and quantified by measuring its absorbance at 260 nm. Its absorbance at 260 nm relative to its absorbance at 280 nm (A260/A280 ratio) fell within the range of values that indicated a pure preparation. An attempt to verify the quantity of DNA using a second independent method specific for DNA, the diaminobenzoic acid dihydrochloride fluorescence assay, revealed a significant discrepancy between the two methods. The difference appeared to result from impurities present within the DNA preparation, despite a A260/A280 ratio that indicated otherwise. We found the A260/A280 ratio to be a poor indicator of the purity of DNA preparations, and determined that significant error may result from quantifying DNA using spectrophotometric methods alone. We propose as an alternative, quantifying DNA using the diaminobenzoic acid dihydrochloride assay in conjunction with uv absorbance at 260 nm and using a FLUOR/A260 ratio as an indicator of DNA purity.
Schy
W E
WE
Institute for Environmental Studies, University of Illinois, Urbana-Champaign 61801.
Plewa
M J
MJ
D062365:Aminobenzoates; D004247:DNA / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D003313:Zea mays
D062365:Aminobenzoates; C017856:3,5-diaminobenzoic acid; D004247:DNA