Melting temperature

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The dissociation of a double-stranded DNA molecule is often referred to as melting because it occurs quickly once a certain temperature has been reached. For multiple copies of a specific DNA molecule (usually synthesized by polymerase chain reaction), the melting temperature (T_m) is defined as the temperature at which 50% of that same DNA molecule species form a stable double helix and the other 50% have been separated to single strand molecules. The melting temperature depends on both the length of the molecule, and the specific nucleotide sequence composition of that molecule.

1 Methods
- 1.1 Wallace method
- 1.2 Nearest-neighbor method
2 References
3 See also
4 External links

[edit] Methods

Several formulas are used to calculate T_m values.^[1]^[2] Some formulas are more accurate in predicting melting temperatures of DNA duplexes.^[3]

[edit] Wallace method

The fastest and less accurate Wallace method is suitable for oligos less than 18mers in length by counting the frequency of each nucleotide base.

T m = 2(A + T) + 4(G + C)

The determination of T_m for DNA extracted from organisms was originally designed to determine the GC content of the DNA using the UV absorbance profile as a function of temperature.^[4]^[5]^[6]

[edit] Nearest-neighbor method

This is the more accurate method used for oligonucleotide melting temperature calculations. Although GC content plays a large factor in the hybridization energy of double-stranded DNA, stacking energy is also a significant contribution. The nearest-neighbor model accounts for this by considering adjacent bases along the backbone two-at-a-time.^[7] Each of these has enthalpic and entropic parameters, the sums of which determine melting temperature according to the following equation:

$T_\mbox{m}=\frac{\Delta H \frac{\mbox{cal}}{\mbox{mol}}}{\Delta S+R \ln(\frac{primer}{2})}-273.15 \ ^\circ \mbox{C}$

where

Δ H

is the enthalpy of base stacking interactions adjusted for helix initiation factors

Δ S

is the entropy of base stacking adjusted for helix initiation factors and for the contributions of salts to the entropy

R

is the universal gas constant $\left (\frac{1.987\mbox{ cal}}{\mbox{mol} \cdot ^\circ \mbox{C}} \right)$

[edit] References

^ Breslauer, K.J. et al. (1986). "Predicting DNA Duplex Stability from the Base Sequence". Proc. Natl. Acad. Sci. USA. 83: 3746-3750. (pdf)
^ Rychlik, W. et al. (1990) Nucleic Acids Res. 18, 6409-6412.
^ Owczarzy R., Vallone P.M., Gallo F.J., Paner T.M., Lane M.J. and Benight A.S (1997). "Predicting sequence-dependent melting stability of short duplex DNA oligomers". Biopolymers 44: 217-239. (pdf)
^ Mandel, M. and J. Marmur (1968) Methods in Enzymology 12 Nucleic Acids B, 195-206. For long chain DNA polymers CsCl density gradient analysis
^ Meselson, M. F. W. Stahl and J. Vinograd (1957) Proc. Natl. Acad. Sci. USA. 43, 581-588 yields values which correlate with the melting temperatures
^ Mandel, M. C., Schildkraut and J. Marmur (1968) Methods in Enzymology 12 Nucleic Acids B: 184-195. Where failures to correlate the buoyant density and the melting temperature arise (as in many bacteriophage DNAs) unusual bases have been found replacing all or part of one of the nucleotides in the genome.
^ John SantaLucia Jr. (1998). "A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics". Proc. Natl. Acad. Sci. USA 95 (4): 1460-5. [1]