Nuclease P1 digestion/high-performance liquid chromatography, a practical method for DNA quantitation

We have developed a practical method for quantifying DNA. The method is practical in two ways. First, a single enzyme is used to digest the DNA to nucleotides that are then quantified by HPLC under ordinary conditions. Second, the method quantifies DNA even when it is impure. In our method, “nuclease P1/HPLC,” the DNA is hydrolyzed by nuclease P1 and the resulting 2′-deoxynucleoside 5′-monophosphates are quantified by HPLC with UV detection. This method was applied to several kinds of genomic DNA in terms of origin and method by which it had been purified. Calf thymus DNA (purified by salt precipitation by the supplier), pig liver DNA (purified by phenolic extraction or by anion-exchange chromatography using a Genomic Tip from Qiagen) and mouse skin DNA (similarly purified) were tested. In some cases a given sample was purified by two of these methods. The values for the amount of DNA by our method were compared with those by three other methods: acid hydrolysis/HPLC (selected as a reference procedure), UV absorbance, and dye binding. Agreement for all DNA samples between the values by our method versus those provided by acid hydrolysis/HPLC was within 10% for amounts of DNA in the 19–54 μg range. In contrast, UV absorbance and the dye-binding assay gave differences up to 30–40% relative to the consistent values furnished by acid hydrolysis and our method. Overall, normalizing the concentrations of the DNA (thymus, liver, skin) by acid hydrolysis/HPLC in 10 samples to values of 1.0 gave the following, relative values and standard deviations: 1.01 ± .07 (nuclease P1/HPLC), 0.8 ± 0.17 (dye binding), and 1.1 ± 0.1 (UV). Since one cannot assume that any sample of DNA is pure, and determining purity of DNA is difficult, then nuclease P1/HPLC or acid hydrolysis/HPLC is recommended rather than the UV absorbance or dye binding for quantifying DNA whenever an accurate value is important.