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 exact 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 the acidification of the solution upon cleavage. We demonstrate here that microgram nucleic acid quantities can be determined when one employs highly active nonspecific nucleases in conjunction with an instrumental setup consisting of a temperature-controlled mixing chamber and miniaturized pH electrodes. Because this method determines the total amount of phosphodiester bonds cleaved, it is independent of the composition or the secondary structure of the nucleic acid and, under certain precautions, represents a simple and robust alternative to optical assays for the determination of either the total nucleic acid concentration or the activity of nucleases in biochemical samples.
Gast
F U
FU
Institut für Biochemie, Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 58, D-35392, Giessen, Germany. frank-ulrich.gast@gmd.de
Pingoud
A
A
D017033:Anabaena / Q000201:enzymology; D001671:Biochemistry / Q000295:instrumentation / Q000379:methods*; D004706:Endodeoxyribonucleases / Q000378:metabolism; D004722:Endoribonucleases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009696:Nucleic Acids / Q000032:analysis*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism
D009696:Nucleic Acids; D011506:Proteins; D004706:Endodeoxyribonucleases; D004722:Endoribonucleases; C021835:Serratia marcescens nuclease