Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy

Sean R. Gallagher1

1 Hoefer Scientific Instruments, San Francisco, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Appendix 3L
DOI:  10.1002/0471142735.ima03ls21
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Reliable quantitation of nanogram and microgram amounts of DNA and RNA in solution is essential to researchers using molecular biology techniques. In addition to the traditional absorbance measurements at 260 nm, two more sensitive fluorescence techniques are presented in this appendix. These three procedures cover a range from 5 to 10 ng DNA/ml to 50mg DNA/ml.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Unit Introduction
  • Basic Protocol: Detection of Nucleic Acids Using Absorption Spectroscopy
  • Alternate Protocol 1: DNA Detection Using the DNA-Binding Fluorochrome H33258
  • Alternate Protocol 2: DNA and RNA Detection with Ethidium Bromide Fluorescence
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol: Detection of Nucleic Acids Using Absorption Spectroscopy

 Materials
  • 1× TNE buffer (see recipe)
  • DNA standard solutions (see recipe)
  • 2 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 H33258

 Additional Materials (also see Basic Protocol)
  • 1 mg/ml fluorochrome H33258 (see recipe)
  • H33258 assay solution (see recipe)
  • DNA standard solutions (see recipe)
  • Fluorometric square glass cuvettes or disposable acrylic cuvettes (Sarstedt #67.755)
  • 0- to 2-ml pipettor
  • 0- to 3-µl sequencing syringe with small-bore tips
  • Dedicated filter fluorometer (Hoefer model TKO100) or scanning fluorescence spectrometer (Shimadzu model RF-5000 or Perkin-Elmer model LS-5B or LS-3B)

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

 Additional Materials (also see Basic Protocol)
  • Ethidium bromide assay solution (see recipe)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •  FigureFigure A.3L.1 Fluorochrome 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 spectrofluorophotometer was used for both assays.
    View Image

Videos

Literature Cited

 Literature Cited
    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, New York.
 Key References
    Labarca, C. and Paigen, K. 1980. See above.

Contains a detailed description of the H33258 fluorometric DNA assay.

GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library