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

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Abstract

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.

Introduction

Quantification of genomic DNA does not have to be exact for many purposes, such as assays in which it is evaluated qualitatively (e.g. DNA sequencing) or in a relative way (e.g. assays for loss of heterozygosity). The opposite is true for quantitative assays of DNA adducts. This is because the amounts of adducts are normalized relative to the amount of DNA in these assays.

In quantitative studies of DNA adducts, the amount of normal DNA usually is determined by UV absorbance, dye binding, 32P-postlabeling, or enzymatic hydrolysis to 2′-deoxyribonucleosides followed by HPLC. The accuracy of an absorbance measurement cannot be trusted since it is based on the unconfirmed assumption that the DNA is pure and entirely in a double-stranded or single-stranded form. While the ratio of absorbance at 260 nm relative to 280 nm often is measured to assess DNA purity, the unreliability of this technique has been pointed out [1]. Dye-binding assays in general are subject to interferences, usually without furnishing any clues as to when interferences are present. 32P-postlabeling for DNA quantification is limited both by its reliance on a radioisotope, and on the assumption, which is not convenient to test, that the yield of radioenzymatic step is 100%. The precision can be poor because the technique is not rugged.

Several hydrolysis/HPLC techniques can be used to overcome the above problems in quantifying DNA. For example, guanine has been measured after acid hydrolysis of DNA [2], [3]. One or more deoxynucleosides have been measured by HPLC after enzymatic hydrolysis [3], [4], [5], and 2′-deoxynucleoside 3′-monophosphates have been measured similarly after enzymatic hydrolysis [6]. However, the use of acidic conditions (which requires a sealed vial and evaporation of acid) or multiple enzymes (two to four) reduces the convenience of these techniques.

Here we present an alternative method for quantifying DNA, involving enzymatic hydrolysis of DNA with a single enzyme to 2′-deoxynucleoside 5′-monophosphates followed by HPLC. We were encouraged to develop and validate this method after we obtained HPLC chromatograms previously that essentially displayed peaks only for mononucleotides when DNA was digested with nuclease P1 [7].

Section snippets

Reagents

Calf thymus DNA was purchased from Worthington (Lakewood, NJ, USA) and Ultra-Pure Calf Thymus DNA, all solvents (HPLC grade), nucleotides and nucleobases were from Sigma–Aldrich (St. Louis, MO, USA). These and the DNA samples that we extracted from tissue samples were dissolved initially in water (e.g. 1 mg/mL) prior to dilution in water or TNE buffer, which was 10 mM Tris, 0.2 M NaCl, 1 mM EDTA, adjusted to pH 7.4 with HCl. Nuclease P1, obtained from Roche Applied Science, Indianapolis, IN, USA as

Results and discussion

The method we selected as a reference procedure to provide an accurate concentration of DNA, even when the DNA is impure, was acid hydrolysis to nucleobases followed by HPLC with UV detection. For this method, the HPLC peaks were calibrated by relying on external standards of the nucleobases, which in turn were measured by absorbance. As described below, the consistent agreement between the results by acid hydrolysis/HPLC and our nuclease P1/HPLC methods added to our confidence in the prior

Conclusion

Digestion with nuclease P1 followed by HPLC is a practical and reliable method for quantifying DNA even when the DNA is isolated from a more difficult tissue sample such as liver or skin. The results from acid hydrolysis/HPLC and nuclease P1/HPLC agreed for a diversity of ten DNA samples varying in degree of purity. In contrast, caution is urged when using a dye-binding or UV absorbance assay to quantify DNA of unknown purity. Probably the sensitivity of this nuclease P1/HPLC method can be

Acknowledgements

The work was supported by NIH Grant CA71993 received as a subcontract from Harvard Medical School, NIH Grants CA84641 and CA 106006, and the Environmental Cancer Research Program. This is contribution No. 877 from the Barnett Institute.

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Present address: Supelco, Bellefonte, PA, USA.

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