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Spectrophotometers

Spectrophotometers. Nucleic Acids and Proteins. 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.

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Spectrophotometers

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  1. Spectrophotometers Nucleic Acids and Proteins

  2. 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.

  3. Molar Extinction Coefficients of Bases

  4. 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.

  5. 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:

  6. 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.

  7. UV radiation can be used to sterilize: the absorbed energy destroys the DNA and kills the organism.

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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

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

  16. 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.

  17. 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:

  18. Amino acid Extinction Coefficient e (lmax) Trp Tryptophan 5,050 M-1cm-1 (280 nm) Tyr Tyrosine 1,440 M-1cm-1 (274 nm) Phe Phenylalanine     220 M-1cm-1 (257 nm)

  19. 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.

  20. Properties of Absorbance and Fluorescence Spectrophotometric Assays for DNA and RNA Absorbance Fluorescence Property (A 260) H33258 EtBr Sensitivity (ug/ml) DNA 1-50 0.01-15 0.1-10 RNA 1-40 n.a. 0.2-10 Ratio DNA/RNA 0.8 400 2.2

  21. 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.

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