Estimation of residual DNA in Feulgen reaction. A new correction of DNA determination per nucleus

Abstract

For the determination of the residual DNA amount after acid hydrolysis of Feulgen's method, a high salt-fluorochrome assay for DNA (5 microM Hoechst 33258 with 1 M NaCl) was effectively applied. At an optimal time length of acid hydrolysis for Feulgen reaction, the ratio of the residual DNA of non-hydrolysis to total DNA is 10% or more in hepatocyte or lymphocyte nuclei. A lot of residual DNA seems not to be negligible in Feulgen's method. A more accurate determination of DNA can be made by correcting the loss ratio of the residual DNA value to Feulgen DNA value. Thus, the combination assay of Feulgen's method with the present fluorometry is enough to measure separately both the amounts of Feulgen DNA and its residual DNA and successfully determines more accurately the total DNA per nucleus by summing both the amounts. The residual DNA, a resistant portion of the chromatin DNA against acid hydrolysis, is a possible constituent as the physiological component of nuclear structures.