Assay for the quantification of intact/fragmented genomic DNA

doi:10.1016/j.ab.2006.07.035

Analytical Biochemistry

Volume 358, Issue 2, 15 November 2006, Pages 247-256

https://doi.org/10.1016/j.ab.2006.07.035 Get rights and content

Abstract

This study shows that the accuracy of the quantification of genomic DNA by the commonly used Hoechst- and PicoGreen-based assays is drastically affected by its degree of fragmentation. Specifically, it was shown that these assays underestimate by 70% the concentration of double-stranded DNA (dsDNA) with sizes less than 23 kb. On the other hand, DNA sizes greater and less than approximately 23 kb are commonly characterized as intact and fragmented genomic DNA, respectively, by the agarose electrophoresis DNA smearing assay and are evaluated only qualitatively by this assay. The need for accurate quantification of fragmented and total genomic DNA, combined with the lack of specific, reliable, and simple quantitative methods, prompted us to develop a Hoechst/PicoGreen-based fluorescent assay that quantifies both types of DNA. This assay addresses these problems, and in its Hoechst and PicoGreen version it accurately quantifies dsDNA as being either intact (⩾23 kb) or fragmented (<23 kb) in concentrations as low as 3 ng ml⁻¹ or 5 pg ml⁻¹ with Hoechst or PicoGreen, respectively, as well as the individual fractions of intact/fragmented DNA existing in any proportions in a total DNA sample in concentrations as low as 10 ng ml⁻¹ or 15 pg ml⁻¹ with Hoechst or PicoGreen, respectively. Because the assay discriminates total genomic DNA in the two size ranges (⩾23 and <23 kb) and quantitates them, it is proposed as the quantitative replacement of the agarose gel electrophoresis genomic DNA smearing assay.

Section snippets

Reagents

Dimethyl sulfoxide (DMSO), Tris–HCl base, ethylenediaminetetraacetic acid (EDTA), and MnCl₂ were obtained from Merck (Darmstadt, Germany). Quant-iT PicoGreen was obtained from Invitrogen–Molecular Probes (Eugene, OR, USA). Hoechst 33258 (bis-benzimide), DNA type I (from calf thymus), highly purified unsheared genomic DNA (from calf thymus), DNase I (from bovine pancreas), bovine serum albumin (BSA), and ethidium bromide were purchased from Sigma–Aldrich (St. Louis, MO, USA). DNA size markers

Results and discussion

Preliminary observations in our laboratory showed that the accurate quantification of dsDNA by the most commonly used Invitrogen–Molecular Probes protocols with the fluorescent dyes Hoechst and PicoGreen [20] depends strongly on its degree of fragmentation, whereas its UV absorbance (at 260 nm) is not affected. These observations are in contrast to the unconsidered effect of dsDNA size on the fluorescence of DNA–dye complexes in the protocols and in the relevant literature [15], [16], [17], [18]

Acknowledgments

This work was funded by the European Social Fund (ESF), Operational Program for Educational and Vocational Training II (EPEAEK II), and particularly the Program IRAKLEITOS. We thank George Kilias and Stephanos Martimianakis (Department of Biology, University of Patras) for their valuable assistance in the gel electrophoresis experiments.

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Assay for the quantification of intact/fragmented genomic DNA

Abstract

Section snippets

Reagents

Results and discussion

Acknowledgments

Exp. Cell Res.

Mutat. Res.

Anal. Biochem.

Free Radic. Biol. Med.

Immunol. Lett.

Anal. Biochem.

Anal. Biochem.

Endogenous DNA damage in humans: A review of quantitative data

Mutagenesis

Free Radicals in Biology and Medicine

Assay for the quantification of small-sized fragmented genomic DNA

Anal. Biochem.

Comparison of chromatin assays for DNA fragmentation evaluation in human sperm

J. Androl.