Large DNA molecules may melt by segments Since the a:t base pair is held together by only two hydrogen bonds, while the g:c pair has three, you might guess that melting temperature was just a function of percent a:t pairs. Generally DNA rich in a:t does have a low melting temperature. Thus, for example, in very long DNA molecules with stretches rich in a:t pairs, and other regions rich in g:c pairs, the a:t stretches will melt before the g:c ones when the temperature is gradually increased. The curve of absorbance versus temperature will then increase slowly, the broad nature of the transition indicating compositional heterogeneity.

				Negative free energy of forming one base pair above another [Breslauer et al., PNAS 83: 3746, 1986]. Values in Table 2 multiplied by 4.184 to convert kcal to Joules and table formated to 4x4. along the top row: the existing base pair				Small DNA molecules melt "all or none" Oligonucleotides 10-40 base pairs long, will have a sharp absorbance-temperature profile indicating an essentially all or nothing denaturation process. The melting temperature, Tm, of molecules with the same average composition, but different sequence, can differ greatly. This sequence dependence is due to the large contribution of base stacking to helix stability, and the dependence of stacking energy on sequence. If sequence were not important, the numbers in each row of the table to the left would be the same. The polarity of the phosphodiester backbone is also important, and two diagonal entries may be different. The biggest dependence on sequence is the 3 fold greater stability of g:c after C :G compared to c:g after A:T.
				A:T T:A C:G G:C a:t 8 4 8 7 t:a 6 8 7 5 c:g 5 7 13 13 g:c 7 8 15 13
		new base pair
					Notation used in table: existing pair is on left; 1st base on top. <-- 1st row, 2nd col   <-- 2nd row, 1st col These are not the same molecules, thus the free energies of pair formation need not be the same.
	5'-T-a-3' 3'-A-t-5' 5'-A-t-3' 3'-T-a-5'

	Salt concentration The phosphate groups are moderately strong acids, and thus they are always ionized at physiological pHs. The two oxygens are equivalent, so they share the single negative charge. These negative charges tend to blow the double helix structure apart, which is perhaps why I haven't included them in the forces that hold the helix together. Why is the helix stable? Inside the cell, the monovalent salt concentration is about 0.15 M. The major cation in the cell is K, while in the diagram I snow Na (no matter). The concentration of positive ions is greater than average around the negative phosphates, and this screens the repulsive force between the phosphates. Since the screen is dependent on the salt, the melting temperature should be dependent on the salt concentration.
		One of the better formulas for melting temperature [Richly and Rhoads, Nucleic Acids Res 17: 8543, 1989] uses the nearest neighbor thermodynamic data, i.e. del H and del S, from the same study quoted at the start of this section (c is the oligonucleotide concentration). Tm = (del H / (del S + R * ln (c/4)))- 273 + 16.6 * log (salt) Thus, for every 10 fold decrease in salt concentration, the Tm decreases about 17 degrees C. This means that most DNA will denature at room temperature (20 C) when the salt concentration falls below 10-5 to 10-6 M.