Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy

Sean R. Gallagher1, Philippe R. Desjardins2

1 UVP, Inc., Upland, California, 2 NanoDrop Technologies, Inc., Wilmington, Delaware
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Appendix 3D
DOI:  10.1002/0471142905.hga03ds53
Online Posting Date:  April, 2007
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Abstract

Quantitation of nucleic acids is a fundamental tool in molecular biology that requires accuracy, reliability, and the use of increasingly smaller sample volumes. This unit describes the traditional absorbance measurement at 260 nm and three more sensitive fluorescence techniques, as well as three microvolume methods that use fiber optic technology in specialized cells or instrumentation. These procedures allow quantitation of DNA solutions ranging from 1 pg/┬Ál to 50 mg/ml.

Keywords: spectroscopy; DNA; RNA; quantitation; microvolume

     
 
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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
  • Alternate Protocol 3: DNA Detection Using Picogreen dsDNA Quantitation Reagent
  • Alternate Protocol 4: Quantitation of Nucleic Acids Using a Microvolume Spectrophotometer Without the Use of Cuvettes or Capillaries
  • Alternate Protocol 5: High‐Sensitivity Quantitation of Nucleic Acids Using a Microvolume Fluorospectrometer Without the Use of Cuvettes or Capillaries
  • Alternate Protocol 6: Quantitation of Nucleic Acids Using a Using a Traditional Spectrophotometer With a Microcell Cuvette
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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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 (Hoefer DQ 300, Turner Biosystems TBS‐380, or Invitrogen Qubit) or scanning fluorescence spectrophotometer (Shimadzu RF‐5301PC or Hitachi F‐2500)
  • 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)

Alternate Protocol 3: DNA Detection Using Picogreen dsDNA Quantitation Reagent

  • PicoGreen dsDNA quantitation kit (Invitrogen) containing:
    • PicoGreen dsDNA quantitation reagent (Component A), 1 ml solution in DMSO (store frozen up to 6 months at −20°C, protected from light)
    • 20× TE (Component B), 25 ml of 200 mM Tris·Cl/20 mM EDTA, pH 7.5 (store up to 6 months at 4°C; may be frozen for long‐term storage)
    • Lambda DNA standard (Component C), 1 ml of 100 µg/ml in TE (store up to 6 months at 4°C; may be frozen for long‐term storage)
  • Spectrofluorometer or fluorescence microplate reader
NOTE: For either the kits or the stand‐alone reagent, sufficient reagent is supplied for 200 assays using an assay volume of 2 ml according to the protocol below. Note that the assay volume is dependent on the instrument used to measure fluorescence; with a microplate reader and a 96‐well microplate, the assay volume is reduced to 200 µl and 2000 assays are possible. The PicoGreen reagent supplied in the kits is exactly the same as the reagent sold separately. The DMSO stock solution should be stored frozen at −20°C and protected from light. The 20× assay buffer and lambda DNA standard in the kits are best stored at 4°C; however, either may be frozen for long‐term storage. When properly stored, components should be stable for at least 6 months.

Alternate Protocol 4: Quantitation of Nucleic Acids Using a Microvolume Spectrophotometer Without the Use of Cuvettes or Capillaries

  Materials
  • Nucleic acid sample to be quantitated
  • Water or buffer in which sample is dissolved
  • NanoDrop ND‐1000 Spectrophotometer (NanoDrop Technologies, Inc., http://www.nanodrop.com)

Alternate Protocol 5: High‐Sensitivity Quantitation of Nucleic Acids Using a Microvolume Fluorospectrometer Without the Use of Cuvettes or Capillaries

  Materials
  • PicoGreen dsDNA Quantitation Kit (Invitrogen) including:
    • 20× TE buffer
    • dsDNA standards
    • PicoGreen dsDNA reagent
  • Nuclease‐free H 2O (e.g., Invitrogen)
  • Unknown samples for assay
  • Nuclease‐free low‐retention pipet tips appropriate for delivering 2 µl (e.g., Hamilton)
  • 1.5‐ml nuclease‐free amber microcentrifuge tubes (e.g., VWR) or standard nuclease‐free microcentrifuge tubes wrapped in aluminum foil
  • NanoDrop ND‐3300 Fluorospectrometer (NanoDrop Technologies, Inc., http://www.nanodrop.com)

Alternate Protocol 6: Quantitation of Nucleic Acids Using a Using a Traditional Spectrophotometer With a Microcell Cuvette

  Materials
  • Optical Ultra LabelGuard Microliter cell (Implen, http://www.implen.de; offered with two basic heights to allow use in a wide variety of spectrophotometers; also available from The Gel Company, http://www.gelcompany.com/); alternative microcell is TrayCell (Hellma; http://www.hellmausa.com/)
  • UV‐Vis spectrophotometer (NanoPhotometer from Implen or any standard or small‐footprint spectrophotometer)
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Figures

Videos

Literature Cited

   Applied Biosystems. 1987. 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.
   Voolstra, C., Jungnickel, A., Borrmann, L., Kirchner, R., and Huber, A. 2006. Spectrophotometric Quantification of Nucleic Acids: LabelGuard enables photometric quantification of submicroliter samples using a standard photometer. Implen Applications Note, Munich, Germany.
   Wallace, R.B. and Miyada, C.G. 1987. Oligonucleotide probes for the screening of recombinant DNA libraries. Methods Enzymol. 152:432‐442.
Key References
   Labarca and Paigen, 1980. See above.
  Contains a detailed description of the Hoechst 33258 fluorometric DNA assay.
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