Analysis of DNA Denaturation

Zbigniew Darzynkiewicz1, Gloria Juan1

1 New York Medical College, Elmsford, New York
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.8
DOI:  10.1002/0471142956.cy0708s03
Online Posting Date:  May, 2001
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Abstract

This unit provides a detailed method for evaluating in situ DNA denaturation by flow cytometry. The principal technique is based upon the metachromatic properties of acridine orange. This technique has a number of advantages over traditional biochemical methods, but requires very precise methodology; the result is excellent clarity and differentiation between single‐stranded and double‐stranded DNA. This unit provides full details on the correct use of acridine orange together with an excellent discussion of the pitfalls and problems.

Keywords: flow cytometry; DNA denaturation; acridine orange; metachromatic dyes; differential staining; single‐stranded DNA; double‐stranded DNA

     
 
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Table of Contents

  • Basic Protocol 1: Differential Staining of Single‐ Versus Double‐Stranded DNA with Acridine Orange
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Differential Staining of Single‐ Versus Double‐Stranded DNA with Acridine Orange

  Materials
  • Cells to be stained
  • PBS, pH 7.4 ( appendix 2A)
  • 70% ethanol, 0° to 4°C
  • 1% formaldehyde in PBS, pH 7.4
  • AO stock solution (see recipe)
  • AO staining solution (see recipe)
  • RNase A solution (see recipe)
  • 0.1 M HCl (preferably stored in an automatic dispensing pipettor bottle set to dispense 0.5 ml)
  • 5‐ml glass or plastic test tubes
  • 15‐ml glass or plastic (preferably polypropylene) centrifuge tubes
  • Clinical centrifuge
  • Ice‐bath hemacytometer
  • Flow cytometer equipped either with 488‐ or 457‐nm argon ion laser or with mercury arc or xenon lamp and BC12 blue filter as fluorescence excitation source, and with appropriate filters (530 ± 30–nm bandpass filter and 640‐nm long‐pass filter for green and red fluorescence, respectively)
  • Additional reagents and equipment for cell culture and trypsinization ( appendix 3B)
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Figures

Videos

Literature Cited

Literature Cited
   Darzynkiewicz, Z. 1994. Acid‐induced denaturation of DNA in situ as a probe of chromatin structure. Methods Cell Biol. 41:527‐541.
   Darzynkiewicz, Z. and Kapuscinski, J. 1990. Acridine orange: A versatile probe of nucleic acids and other cell constituents. In Flow Cytometry and Sorting (M.R. Melamed, T. Lindmo, and M.L. Mendelsohn, eds.) pp.291‐314. Wiley‐Liss, New York.
   Darzynkiewicz, Z., Traganos, F., Sharpless, T., and Melamed, M.R. 1977. Different sensitivity of DNA in situ in interphase and metaphase chromatin to heat denaturation. J. Cell Biol. 73:128‐138.
   Darzynkiewicz, Z., Traganos, F., Sharpless, T., and Melamed, M.R. 1976. Lymphocyte stimulation: A rapid multiparameter analysis. Proc. Natl. Acad. Sci. U.S.A. 73:2881‐2884.
   Darzynkiewicz, Z., Traganos, F., and Kimmel, M. 1987. Assay of cell cycle kinetics by multivariate flow cytometry using the principle of stathmokinesis. In Techniques in Cell Cycle Analysis (J.W. Gray and Z. Darzynkiewicz, eds.) pp.291‐336. Humana Press, Clifton, N.J.
   Darzynkiewicz, Z., Juan, G., Li, X., Gorczyca, W., Murakami, T., and Traganos, F. 1997. Cytometry in cell necrobiology: Analysis of apoptosis and accidental cell death (necrosis). Cytometry 27:1‐20.
   Evenson, D.P., Darzynkiewicz, Z., and Melamed, M.R. 1980. Relation of mammalian sperm chromatin heterogeneity to fertility. Science 210:1131‐1133.
   Frankfurt, O.S., Byrnes, J.J., Seckinger, D., and Sugarbaker, E.V. 1993. Apoptosis (programmed cell death) and the evaluation of chemosensitivity in chronic lymphocytic leukemia and lymphoma. Onc. Res. 5:37‐42.
   Hotz, M.A., Traganos, F., and Darzynkiewicz, Z. 1992. Changes in nuclear chromatin related to apoptosis or necrosis induced by the DNA topoisomerase II inhibitor fostriecin in MOLT‐4 and HL‐60 cells are revealed by altered DNA sensitivity to denaturation. Exp. Cell Res. 201:184‐192.
   Rigler, R., Jr. 1966. Microfluorometric characterization of intranuclear nucleic acids and nucleoproteins by acridine orange. Acta Physiol. Scand. 67(Suppl. 267):1‐122.
   Subirana, J.A. 1973. Studies on the thermal denaturation of nucleohistone. J. Mol. Biol. 74:363‐385.
   van Holde, K.E. 1989. Chromatin. Springer‐ Verlag, New York.
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