User Ratings

Easy to Follow Achieved Expected Results Overall Rating Add your comments

Analysis of DNA Denaturation

Zbigniew Darzynkiewicz¹, Gloria Juan¹

¹New 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

GO TO THE FULL TEXT:

PDF or HTML at Wiley Online Library

Are you the author of this protocol? Login or register and return to this page.

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

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Unit Introduction
Basic Protocol: Differential Staining of Single- Versus Double-Stranded DNA with Acridine Orange
Reagents and Solutions
Commentary
Literature Cited
Figures

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Materials

Basic Protocol: 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)

Looking for materials? Suppliers Guide

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Figures

Figure 7.8.1

Characteristic AO staining pattern of exponentially growing Friend erythroleukemia cells after incubation with RNase and partial DNA denaturation by HCl. The cells were processed and stained as described in Basic Protocol. The left panel shows cell distribution with respect to red versus green fluorescence; the right panel represents the total (red plus green) fluorescence versus _t, where _t represents the ratio of red to total fluorescence. G₁ cells may divide into A and B subpopulations, as shown (dashed skewed line). The DNA of early G₁ (G_1A) postmitotic cells is more denatured (higher _t) than that of late G₁ (G_1B) cells. Mitotic (M) cells have the highest _t and can easily be distinguished from G₂ cells.

View Image
Figure 7.8.2

Changes in stainability with AO of human peripheral blood lymphocytes mitogenically stimulated with phytohemagglutinin (PHA). (A) Unstimulated cells. (B) Cells stimulated with PHA for 18 hr. (C) Cells stimulated for 3 days. (D) Cells stimulated for 3 days, with vinblastine included in the culture for the final 6 hr to cause mitotic arrest. Cells were stained as described in Basic Protocol. Transition of cells from G₀ to G₁ is associated with an increase in green fluorescence and a decrease in red—i.e., a decrease in DNA denaturability (cells in transition T, panel B). A population of mitotic cells (M) becomes apparent in the vinblastine-treated culture.

View Image
Figure 7.8.3

Detection of apoptotic cells based on differences in DNA sensitivity to denaturation. Total fluorescence is on the y axis. (A) Exponentially growing HL-60 promyelocytic leukemia cells (control). (B,C) Cells treated in vitro with 0.15 µM camptothecin (CPT) for 2 (B) and 3 hr (C) to induce apoptosis. Cells were stained as described in Basic Protocol. Note that in the control only mitotic (M) cells have high _t. The S-phase cells undergoing apoptosis (Ap) in the presence of CPT are characterized by increased DNA denaturability.

View Image

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.