Nucleic acid quantification by delta pH measurement

Nucleic acids are quantitated by UV absorbance measurement, fluorimetry, or hybridization. While the latter method is time-consuming and requires exac t knowledge of the sequence, spectroscopic methods require that the sample does not contain UV-absorbing or fluorescent material. An enzymatic method is the measurement of the hyperchromic change upon cleavage of the nucleic acids by nucleases (Kunitz assay). A variation of this assay makes use of t he acidification of the solution upon cleavage. We demonstrate here that mi crogram nucleic acid quantities can be determined when one employs highly a ctive nonspecific nucleases in conjunction with an instrumental setup consi sting of a temperature-controlled mixing chamber and miniaturized pH electr odes. Because this method determines the total amount of phosphodiester bon ds cleaved, it is independent of the composition or the secondary structure of the nucleic acid and, under certain precautions, represents a simple an d robust alternative to optical assays for the determination of either the total nucleic acid concentration or the activity of nucleases in biochemica l samples. (C) 2001 Elsevier Science B.V. All rights reserved.