Spectroscopic Analysis Using DNA and RNA Fluorescent Probes

Sean R. Ghallagher1

1 Motorola Corporation, Tempe, Arizona
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 4.5
DOI:  10.1002/0471142956.cy0405s14
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The unit takes the nature of fluorescent probes one step further and provides a quantitative approach to analysis of DNA and RNA probes using spectroscopic techniques. Two sensitive fluorescence protocols are provided in addition to the traditional absorbance method.

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

Table of Contents

  • Basic Protocol 1: 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
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: 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

  • 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

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

Figures

Videos

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. Mol. Biol. 5:109‐118.
   Portugal, J. and Waring, M.J. 1988. Assignment of DNA binding sites for 4′,6,diamdine‐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 recominant DNA libraries. In Methods of Enzymology Vol. 152: 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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library