Determination of DNA by sub- and super-equivalence method of isotope dilution analysis using enzyme reaction

Sub- and super-equivalence method of isotope dilution analysis (SSE-IDA) using enzyme reaction was first applied for the determination of biological substance, DNA. Radioactive DNA (pUC18) to be analysed was prepared by incorporating 3H-thymidine in growing E. coli. A part of DNA was cut into the linear form (L-form) DNA under definite conditions using a restriction enzyme HindIII, following the separation of each by gel electrophoresis. Radioactivites of separated L-form DNA of two series were measured. The quantity of DNA was obtained by a graph method of SSE-IDA. As preliminary experiments, it was examined under what conditions the enzyme reaction proceeds as zeroth or first order reaction. We used the enzyme and substrate concentrations near zeroth order, where ordinary subst-IDA seems not to give a satisfactory results. As the results, 0.25 microgram of DNA was determined the error of about 10%.