Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy

Sean R. Gallagher1

1 Motorola Corporation, Tempe, Arizona
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Appendix 3D
DOI:  10.1002/0471143030.cba03ds07
Online Posting Date:  May, 2001
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Abstract

Reliable quantitation of nanogram and microgram amounts of DNA and RNA in solution is essential to researchers in cell and molecular biology. In addition to the traditional absorbance measurements at 260 nm, two more sensitive fluorescence techniques–Hoescht 33258 dye binding, ethidium bromide binding are described. These three procedures cover a range from 5 to 10 ng/ml DNA to 50m g/ml DNA.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Detection of Nucleic Acids Using Absorption Spectroscopy
  • Alternate Protocol 1: DNA Detection Using the DNA-Binding Fluorochrome Hoechst 33258
  • Alternate Protocol 2: DNA and RNA Detection with Ethidium Bromide Fluorescence
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Figures
  • Tables
     
 
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Materials

Basic Protocol: Detection of Nucleic Acids Using Absorption Spectroscopy

 Materials
  • 1× TNE buffer (see recipe)
  • DNA sample to be quantitated
  • Calf thymus DNA standard solutions (see recipe)
  • Matched quartz semi-micro spectrophotometer cuvettes (1-cm pathlength)
  • Single- or dual-beam spectrophotometer (ultraviolet to visible)

Alternate Protocol 1: DNA Detection Using the DNA-Binding Fluorochrome Hoechst 33258

 Additional Materials (also see Basic Protocol)
  • Hoechst 33258 assay solution (working solution; see recipe)
  • Dedicated filter fluorometer (e.g., Amersham Pharmacia Biotech DQLOO) or scanning fluorescence spectrophotometer (e.g., Shimadzu model RF-5000 or Perkin-Elmer model LS-5B or LS-3B)
  • Fluorometric square glass cuvettes or disposable acrylic cuvettes (Sarstedt)
  • Teflon stir rod

Alternate Protocol 2: DNA and RNA Detection with Ethidium Bromide Fluorescence

 Additional Materials (also see Basic Protocol)
  • Ethidium bromide assay solution (see recipe)
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Figures

  •  FigureFigure A.3D.1 Fluorochrome Hoechst 33258 (H33258) (A) and ethidium bromide (B) DNA concentration standard curves. Assays were performed as described in alternate protocols, at indicated excitation and emission wavelengths. The concentrations of H33258 and ethidium bromide were 0.1 and 5 µg/ml, respectively. Assays contained the indicated concentrations of calf thymus DNA standards suspended in a final volume of 2.0 ml. Inset shows low DNA concentration curve for the H33258 assay. Note that, under these conditions, H33258 produces ~20 times more relative fluorescence units than ethidium bromide. A Shimadzu RF-5000 scanning fluorescence spectrometer was used for both assays.
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Literature Cited

 Literature Cited
    Applied Biosystems. 1989. User Bulletin Issue 11, Model No. 370. Applied Biosystems, Foster City, Calif.
    Cesarone, C.F., Bolognesi, C., and Santi, L. 1979. Improved microfluorometric DNA determination in biological material using 33258 Hoechst. Anal. Biochem. 100:188-197.
    Daxhelet, G.A., Coene, M.M., Hoet, P.P., and Cocito, C.G. 1989. Spectrofluorometry of dyes with DNAs of different base composition and conformation. Anal. Biochem. 179:401-403.
    Labarca, C. and Paigen, K. 1980. A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem. 102:344-352.
    Le Pecq, J.-B. 1971. Use of ethidium bromide for separation and determination of nucleic acids of various conformational forms and measurement of their associated enzymes. In Methods of Biochemical Analysis, Vol. 20 (D. Glick, ed.) pp. 41-86. John Wiley & Sons, New York.
    Marmur, J. and Doty, P. 1962. Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J. Molec. Biol. 5:109-118.
    Portugal, J. and Waring, M.J. 1988. Assignment of DNA binding sites for 4¢,6-diamidine-2- phenylindole and bisbenzimide (Hoechst 33258): A comparative footprinting study. Biochem. Biophys. Acta 949:158-168.
    Stout, D.L. and Becker, F.F. 1982. Fluorometric quantitation of single-stranded DNA: A method applicable to the technique of alkaline elution. Anal. Biochem. 127:302-307.
    Van Lancker, M. and Gheyssens, L.C. 1986. A comparison of four frequently used assays for quantitative determination of DNA. Anal. Lett. 19:615-623.
    Wallace, R.B. and Miyada, C.G. 1987. Oligonucleotide probes for the screening of recombinant DNA libraries. In Methods of Enzymology, Vol. 152: Guide to Molecular Cloning Techniques (S.L. Berger and A.R. Kimmel, eds.) pp. 432-442. Academic Press, San Diego.
 Key References
    Labarca and Paigen, 1980. See above

Contains a detailed description of the Hoechst 33258 fluorometric DNA assay.

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