20.309: Biological Instrumentation and Measurement
DNA in solution
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Consider a solution containing equal quantities of complementary single stranded DNA (ssDNA) oligonucleotides <math>\left . A \right .</math> and <math>\left . A' \right .</math>.

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Complementary ssDNA strands bond to form double stranded DNA (dsDNA). The reaction is governed by the equation <math>1 A + 1 A' \Leftrightarrow 1 A \cdot A'</math>

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The forward reaction in which two ssDNA oligos combine to form dsDNA is called annealing. The reverse process is called thermal denaturation or melting.

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At low temperatures, dsDNA is favored. As the temperature rises, dsDNA increasingly separates into its component ssDNA oligos. (Think about why with respect to enthalpic and entropic considerations.)

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The melting temperature, <math>\left . T_m \right .</math>, is defined to be the point where half of the dsDNA is denatured.

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Short sequences of about 1040 base pairs (such as those used in the DNA Melting lab) tend to denature all at once. Longer sequences may melt in segments.

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Less energy is required to split the double hydrogen bond of AT pairs than the triple bond of GC pairs. Thus, AT rich sequences tend to melt at lower temperatures than GC rich ones.^{[1]}

Several web tools are available to predict the melting temprature. (See, for example, DINA Melt or Oligocalc.)
Fundamental equilibrium relationships
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The value of <math>\left . K_{eq} \right .</math> is a function of temperature. We can equate the fundamental definition of the standard free energy change with its relationship to the equilibrium constant in solution:

 <math>
\begin{align}
\Delta G^{\circ} & = \Delta H^{\circ}  T \Delta S^{\circ}\\
& = R T \ln K_{eq}\\
\end{align}
</math>
 where
 <math>\Delta G^{\circ}</math> is the standard change in free energy
 <math>\Delta H^{\circ}</math> is the standard enthalpy change
 <math>\left . T \right .</math> is the temperature
 <math>\Delta S^{\circ}</math> is the standard entropy change
 <math>\left . R \right .</math> is the gas constant
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Solving for <math>\left . K \right .</math>:

 <math>
K_{eq} = e^\left [\frac{\Delta S^{\circ}}{R}  \frac{\Delta H^{\circ}}{R T} \right ] \quad (1)
</math>
Note that the above equation can be differentiated with respect to temperature to yield the (perhaps once!) familiar van't Hoff equation.
Theoretical relation of dsDNA fraction and thermodynamic parameters
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In the lab, the fraction of dsDNA will be measured with a fluorescent dye that preferentially binds to dsDNA. As such, it will be useful to derive an equation that relates the fraction of dsDNA to temperature and the thermodynamic parameters.

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Let <math>\left . f \right .</math> be the fraction of total DNA that is double stranded

 <math>
f = \frac{2 C_{DS}}{C_T} = \frac{C_T  2 C_{SS}}{C_T} = 1  2 \frac{C_{SS}}{C_T}
</math>
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{{{1}}}

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Now we can solve for <math>\left . K \right .</math> in terms of <math>\left . f \right .</math> and <math>\left . C_T \right .</math>:

 <math>
K_{eq} = \frac{C_{DS}}{C_{SS}^2}
= \frac{f C_T / 2}{ [(1  f) C_T / 2] ^ 2}
= \frac{2 f}{(1  f)^2 C_T}
</math>
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{{{1}}}

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Substituting for K_{eq} from equation 1:

 <math>
e^\left [\frac{\Delta S}{R}  \frac{\Delta H}{R T} \right ] = \frac{2 f}{(1  f)^2 C_T} \quad (2)
</math>
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Taking the log of both sides and solving for <math>\left . T \right .</math>:

 <math>
T(f) = \frac{\Delta H^{\circ}}{\Delta S^{\circ}R \ln(2f/C_T(1f)^2)}
</math>
We now know temperature as a function of dsDNA fraction for a given total DNA strand concentration and DNA identity (therefore ΔH, etc.).
Simulating DNA melting for tractable calculation of dsDNA fraction
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For simulating DNA melting experiments, it will be convenient to have an expression for <math>\left . f \right .</math> in terms of <math>\left . T \right .</math>. Unfortunately, this gets pretty yucky. On the bright side, Matlab and Python are good at calculating yuck.

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Taking the log of both sides of equation 2 (after resubstiting in equation 1 for simplicity) and using the quadratic formula (eliminating the nonphysical root):

 <math>
f = \frac{1 + C_T K_{eq}  \sqrt{1 + 2 C_T K_{eq}}}{C_T K_{eq}}
</math>
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Substituting from equation 1 gives the desired result.

 <math>
f = \frac{1 + C_T e^\left [\frac{\Delta S^{\circ}}{R}  \frac{\Delta H^{\circ}}{R T} \right ]  \sqrt{1 + 2 C_T e^\left [\frac{\Delta S^{\circ}}{R}  \frac{\Delta H^{\circ}}{R T} \right ]}}{C_T e^\left [\frac{\Delta S^{\circ}}{R}  \frac{\Delta H^{\circ}}{R T} \right ]}
</math>
See the pages DNA Melting Part 1: Simulating DNA Melting  Basics. And if you're interested in a Python implementation see Python:Simulating DNA Melting
References
 ↑ Breslauer et al., Predicting DNA duplex stability from the base sequence PNAS 83: 3746, 1986