Optical Methods

G. Eric Plum1

1 Rutgers University, Piscataway, New Jersey
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 7.3
DOI:  10.1002/0471142700.nc0703s00
Online Posting Date:  May, 2001
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Abstract

Equilibrium optical melting curves are used in this unit to determine thermodynamic parameters of nucleic acid complex formation. This section contains all of the relevant equations and a discussion of which are most appropriate to a given situation. Additionally, procedures are given for making preliminary determinations of molar extinction coefficients and for determining the number of oligonucleotides in a complex. The section on extinction coefficients is particularly essential to anyone needing to know solution concentrations.

     
 
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Table of Contents

  • Basic Protocol 1: Determination of Oligonucleotide Molar Extinction Coefficients
  • Basic Protocol 2: Determination of Molecularity and Extinction Coefficients of Oligonucleotide Complexes
  • Basic Protocol 3: Preparation of Equilibrium Melting Curves
  • Support Protocol 1: Calculation of ΔH° from Concentration‐Dependent Melting Curves
  • Support Protocol 2: Calculation of ΔH° from α(T) Versus T Plots
  • Support Protocol 3: Calculation of ΔH° by Direct Application of the Van't Hoff Equation
  • Support Protocol 4: Calculation of ΔH° from Differential Curves
  • Support Protocol 5: Calculation of ΔG° Using the Van't Hoff Equation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Determination of Oligonucleotide Molar Extinction Coefficients

  Materials
  • 10 mM cacodylate buffer solution (see recipe)
  • 1 mg/mL nuclease P1 solution (see recipe)
  • 100 U/mL alkaline phosphatase solution (see recipe)
  • Oligonucleotide to be analyzed
  • 53 µM poly(rU) (Amersham Pharmacia Biotech) in 10 mM cacodylate buffer solution recipe
  • Standard phosphate solution (see recipe)
  • ANS solution (see recipe)
  • Molybdate solution (see recipe)
  • 1‐dram screw‐capped glass vials
  • 95°C water bath
  • Single‐ or double‐beam UV/visible spectrophotometer
  • Matched quartz semimicro 10‐mm spectrophotometer cuvettes
NOTE: In all steps, samples should be prepared in 1‐dram screw‐capped glass vials. Plastic microcentrifuge tubes should not be used.

Basic Protocol 2: Determination of Molecularity and Extinction Coefficients of Oligonucleotide Complexes

  Materials
  • Two oligonucleotides, A and B, which are expected to form a complex
  • Buffer
  • Single‐ or double‐beam UV/visible spectrophotometer
  • Quartz semimicro spectrophotometer cuvette (1‐cm pathlength)
  • Additional reagents and equipment for determining molar extinction coefficients (see protocol 1)

Basic Protocol 3: Preparation of Equilibrium Melting Curves

  Materials
  • Oligonucleotides in buffer solution (single folded chains or multimolecular complexes)
  • Double‐beam UV/visible spectrophotometer with temperature‐controlled (stepping and/or scanning) cell holder
  • Stoppered quartz spectrophotometer cuvettes (0.1‐, 0.2‐, 0.5‐, and 1.0‐cm pathlengths) with metal spacers (shorter pathlength cuvettes are useful, if available)
  • Additional reagents and equipment for determining molar extinction coefficients (see protocol 1) and determining molecularity and extinction coefficients of complexes (see protocol 2)
NOTE: Buffers with large heats of ionization, which includes most popular biochemical buffers, should be avoided, as the pH of solutions prepared with these buffers is temperature dependent. Tris solutions are particularly susceptible to this effect. Phosphate, cacodylate, and PIPES are acceptable choices.
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Figures

Videos

Literature Cited

Literature Cited
   Felsenfeld, G., Davies, D.R., and Rich, A. 1957. Formation of a three‐stranded polynucleotide molecule. J. Am. Chem. Soc. 79:2023‐2024.
   Griswold, B.L., Humoller, F.L., and McIntyre, A.R. 1951. Inorganic phosphates and phosphate esters in tissue extracts. Anal. Chem. 23:192‐194.
   Marky, L.A. and Breslauer, K.J. 1987. Calculating thermodynamic data for transitions of any molecularity from equilibrium melting curves. Biopolymers 26:1601‐1620.
   Plum, G.E., Grollman, A.P., Johnson, F., and Breslauer, K.J. 1995a. Influence of the oxidatively damaged adduct 8‐oxodeoxyguanosine on the conformation, energetics, and thermodynamic stability of a DNA duplex. Biochemistry 34:16148‐16160.
   Plum, G.E., Pilch, D.S., Singleton, S.F., and Breslauer, K.J. 1995b. Nucleic acid hybridization: Triplex stability and energetics. Annu. Rev. Biophys. Biomol. Struct. Dyn. 24:319‐350.
   Plum, G.E., Breslauer, K.J., and Roberts, R.W. 1999. Thermodynamics and kinetics of nucleic acid association/dissociation and folding processes. In Comprehensive Natural Products Chemistry, Vol. 7 (E.T. Kool, ed.), pp. 15‐53. Elsevier Science Ltd., Oxford.
   Poland, D. 1974. Recursion relation generation of probability profiles for specific‐sequence macromolecules with long range interactions. Biopolymers 13:1859‐1871.
   Puglisi, J.D. and Tinoco, I. Jr. 1989. Absorbance melting curves of RNA. Methods Enzymol. 180:304‐325.
   Savitsky, A. and Golay, M.J.E. 1964. Smoothing and differentiation of data by simplified least squares procedures. Anal. Chem. 36:1627‐39.
   Snell, F.D. and Snell, C.T. 1949. Phosphorous. In Colorimetric Methods of Analysis, 3rd ed., Vol. 2 pp. 630‐681. Van Nostrand, New York.
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