Nuclease P1 digestion/high-performance liquid chromatography, a practical method for DNA quantitation
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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2012, Bioorganic and Medicinal ChemistryCitation Excerpt :95% A → 50% A over 25 min, 50% A for 5 min, flow rate 40 μL/min). Solutions of PNU with both AT and TA oligomers (R = [drug]/[DNA] = 4.0) were digested as previously described.25 Enzymatic digestion was done on DNA solution 30 μM using nuclease P1 2 U/mL (Sigma–Aldrich, St. Louis, US) in 20 mM ammonium acetate buffer (pH 5.5 and 6.8) with 500 μM ZnCl2.
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Present address: Supelco, Bellefonte, PA, USA.