Spectrophotometers

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Spectrophotometers

• Nucleic Acids and Proteins

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Nucleic Acids and Spectrophotometer

• The rings of the bases (A, C, G, T, U) are made
  up of alternating single and double bonds.
• Such ring structures absorb in the U.V.
• Each of the four nucleotide bases has a slightly
  different absorption spectrum, and the spectrum
  of DNA is the average of them.

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Molar Extinction Coefficients of Bases
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DNA and Spectrophotometers

• Double stranded DNA is a dynamic structure and
  not a static entity.
• The two strands are held together by non-covalent
  interactions (hydrogen bonding and base
  stacking).
• The energy of these interactions allows the
  helix to come apart quite easily at physiological
  temperatures.

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Double-Stranded vs. Single-Stranded Nucleic Acids

• DNA can be heated and, at a certain temperature,
  the two strands will come apart. We say that the
  DNA helix has melted or denatured.
• This transition can be followed by the increase
  in the absorption of ultraviolet light by the
  molecule as it goes from helix to random coil
  (the denatured form). This is called
  hyperchromicity

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Bases Are Exposed in SS DNA

• When a DNA helix is denatured to become single
  strands the absorbance is increased about 30
  percent.
• This increase, (the hyperchromic shift) indicates
  that the double-stranded molecule is quenching
  fluorescence.
• So, you always need to know if your DNA is double
  or single stranded when measuring it using the
  spectrophotometer.

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UV radiation can be used to sterilize the
absorbed energy destroys the DNA and kills the
organism.
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Spectrophotometer and Nucleic Acid Applications

• Measure the complexity of double stranded DNA
  (COT analysis)
• Measure the concentration of double stranded or
  single stranded DNAs, nucleotide mixes and RNA in
  solution.
• Measure how clean the DNA/RNA is relative to
  contaminating protein.

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Molar Extinction Coefficient

• For solution concentrations given in mol/liter
  and a cuvette of 1-cm path length, E is the molar
  extinction coefficient and has units of
• M-1cm-1.
• If concentration units of ug/ml are used, then E
  is the specific absorption coefficient and has
  units of (ug/ml)-1cm-1.

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Molar Extinction Coefficient

• The values of E used here are as follows
• ssDNA, 0.027 (ug/ml)-1cm-1
• dsDNA, 0.020 (ug/ml)-1cm-1
• ssRNA, 0.025 (ug/ml)-1cm-1

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A Convenient Number to Remember

• Using these calculations, an A260 of 1.0
  indicates
• 50 ug/ml double-stranded DNA
• 37 ug/ml single-stranded DNA
• 40 ug/ml single- stranded RNA

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UV Quantitation of DNA

• The detection limit of absorption spectroscopy
  will depend on the sensitivity of the
  spectrophotometer and any UV-absorbing
  contaminants that might be present.
• The lower limit is generally 0.5 to 1 ug nucleic
  acid.

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Practical Use

• Measure some DS DNA in the Spec.
• Absorbance is too high.
• Dilute the DNA to get a reading in range. Keep
  track of the dilution factor (Example 100 ul
  diluted in 900 ul is a 1/10 dilution with a
  dilution factor of 10).
• Multiply 50 X 10 X OD concentration in ug/ml.

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Practical Use

• Dilute some dsDNA 1/100 (dilution factor of 100).
• Absorbance at 260 nm is 0.400.
• 100 X 0.4 X 50 2000 ug/ml
• What if it was RNA?
• 100 X 0.4 X 40 1600 ug/ml

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Practical Use

• If you have 100 ul of DNA of concentration at
  2000 ug/ml, how much RNA do you have?

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Protein Contamination

• Proteins in general have A280 readings
  considerably lower than nucleic acids on an
  equivalent weight basis.
• Thus, even a small increase in the A280 relative
  to A260 (Or a lowering of the A260/A280 ratio)
  can indicate severe protein contamination.

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Spectral Properties of Amino Acids

• Trp, Tyr, and Phe contain conjugated aromatic
  rings.
• Consequently, they absorb light in the
  ultraviolet range (UV).
• The extinction coefficients (or molar absorption
  coefficients) of these three amino acids are

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Proteins and Spectrophotometer

• Trp absorbs UV light the strongest.
• Furthermore, since both Trp and Tyr show the
  maximum light absorbance at approximately 280 nm
  the absorption maximum of most proteins is around
  280 nm.
• In contrast, the absorption maximum for nucleic
  acids is approximately 260 nm.

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Fluorescent Quantitation of DNA

• For the Hoechst 33258 and ethidium bromide
  assays, a plot of relative fluorescence units or
  estimated concentration (y axis) versus actual
  concentration (x axis) typically produces a
  linear regression with a correlation coefficient
  (r2) of 0.98 to 0.99.
• Be certain that the final amount of DNA does not
  exceed the linear portion of the assay.