Factors influencing the determination of dNA with indole. II. Recovery of hydrolyzed dNA from protein precipitates

DNA can be measured by the indole method in hydrolyzates containing large concentrations of nonspecific chromogen without exceeding the sufficiency of the color reagents or the efficiency of the chloroform extraction procedure. Since TCA is nonessential for the completeness of the color reaction, there is considerable latitude in the choice of extraction procedures. The action of hot acid hydrolysis to release the indole chromogen from DNA is opposed by two major factors: (1) the capacity of the hot hydrolysis to entrap solubilized but incompletely hydrolyzed DNA in a protein precipitate and (2) the ability of the hydrolysis to destroy the chromogen for the reaction. Because of these factors and since the rate of chromogen release is eventually exceeded by the rate of its destruction, the hydrolysis should be run with internal and external DNA standards and not exceed 20, 40, 50, or 120 min at 90, 85, 80, or 70°C, respectively. If the recovery of internal standard indicates that regnificant amounts of DNA have been left unmeasured, an increase in the volume of the system is preferable to an increase in the strength of the acid or the number of extractions.