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Melting Temperatures of BacterialGenomic DNA

This experiment is divided into two parts:

1) Isolation of genomic DNA from Escherichia coli and Staphylococcus aureus

See [PROTOCOL FOR GENOMIC DNA extraction for further details]. This has been performed in the interests of time for you.

2) Measuring the melting temperature of these genomes

1) Isolation of DNA

The isolation of DNA is for you in the interest of time. Please go through the protocol (Appendix I).

2) Melting curve measurement

Principle

DNA stability is dependent on the energy of association of the bases. By heating DNA we can monitor its denaturation. For a given sequence of DNA the denaturation curve is charachteristic. And the half-maximum of the transition from dsDNA (double stranded) to ssDNA (single stranded) is referred to as the melting temperature (Tm).

Spectrophotometry in the UV range is a convenient measure of this transition of DNA from a ds to ss form.

We use a nanodrop since it reduces sample volume for absorbance measurement. This device uses surface-tension to spread a drop ~1 μl in volume across a sensor (Thermo Fisher Nanodrop Manual).

Materials

Instruments

1. UV/Vis spectrophotometer (Nanodrop device to be used for small volumes)

2. Heating block (40-95°C).

3. Micro-pipettes with clean sterile tips

Chemicals and Biochemicals
  1. An aqueous buffer containing 10 mM Na2HPO4/NaH2PO4, 1 mM Na2EDTA, pH 7.00 ±0.01
  2. Purified dsDNA from Escherichia coli (conc. 0.15 μg/μl) and Staphylococcus aureus (conc. (conc. 0.84 μg/μl))
  3. 1M MgCl2 as a perturbant (stock)
  4. 5M NaCl as perturbant (stock)
Glass/Plasticware
  1. Plastic tips for micropipettes
  2. Microcentrifuge tubes (eppendorf) for DNA melting

Method

  1. For a single reaction use a final concentration of 2 _g of DNA in 100 _l buffer volume. To prepare this dilute the stock DNA (E. coli , S. aureus) in a total volume of 100 _l using a prepared solution of 10 mM Na2HPO4/NaH2PO4, 1 mM Na2EDTA, dilute 1:5 the stock NaCl (5M stock). ONLY IF you are USING MgCl2 as a perturbant, use a FINAL concentration of 0.1 M MgCl2. make up the volume with deionised water
  2. Concentrations of all DNA solutions will be determined by UV spectrophotometry. Absolute concentrations can be calculated using the extinction coefficients of calf Thymus DNA where ε260 = 12824 M (base pairs)^-1 cm^-1.
  3. Ultraviolet DNA melting curves are determined using a UV/Visible Spectrophotometer.
  4. Temperature control is achieved by placing the tube containing your DNA sample in heating block.
  5. Heating runs are performed between 40 and 95°C. Measure absorbance every 5°C at 260 nm.
  6. The device scales for a 1 cm path length. Measurement of DNA absorbance to be done taking a 1_l droplet.
  7. Ensure you clean the nano-drop before and after your sample is measured.
  8. Melting temperatures are determined from the primary data by calculating the normalized absorbance at 260 nm using the expression A n = A ss − A ( T ) A ss − A h where Ass: absorbance of single stranded DNA (highest temperature), A(T): absorbance at a given temperature T, Ah: absorbance of helical DNA (lowest temperature).
  9. Repeat the heating cycle on the same sample and measure absorbances within the same temperature range as before.
  10. Using MATLAB, Octave, Scilab or Python (Pylab), plot the normalized Absorbance against temperature. Calculate the first derivative of this absorbance and superimpose it on the graph of normalized absorbance. Use the numerical approximation of dA/dT as
  11. Find the X-intercept of the maximal value in the derivative graph of relative absorbance to determine the melting temperature (Tm).
  12. A second exercise for simulation of the melting curve will follow.

    13. Results

    1. Prepare an observations table with Temperature (°C) and Absorbance at 260 nm.

    2. Prepare 3 graphs

    (i) absorbance vs. temperature

    (ii) normalized absorbance vs. temperature

    (iii) First derivative of absorbance vs. temperature

    2. Make a table of the Tm value obtained by you.

    3. Add values from Tm obtained by others (in a separate column) with/without perturbant for the DNA from your organism. Compare to Tm for the DNA from the other species.

    Discussion

    1.What is the result of adding a salt MgCl2 to the DNA sample? Why?

    2.Is there a difference between Average Tm values of E. coli and S. aureus DNA? Why?

    References

    1. Peter M. Murphy , Victoria A. Phillips , Sharon A. Jennings , Nichola C.Garbett , Jonathan B. Chaires , Terence C. Jenkins and Richard T. Wheelhouse(2003) Biarylpyrimidines: a new class of ligand for high-order DNA recognition.Chem. Commun. 1160-1161.
    2. W. David Wilson, Farial A. Tanious, Maria Fernandez-Saiz, C. Ted Rigl (1997) Evaluation of Drug-Nucleic Acid Interactions by Thermal Melting Curves. Meth. Mol. Biol. 90: 219-240
    3. M. Mandel and J. Marmur (1968) Use of ultraviolet absorbance-temperatureprofile for determining the guanine plus cytosine content of DNA. Meth.Enzymol. 12(B) 195-206.
    4. ADT Bio. DNA stability www.adtbio.com
    5. IDT DNA Biophysics biophysics.idtdna.com