USING EXCEL TO DETERMINE THE MELTING POINT (TM) OF DNA
From your experiment you will have two columns of data: The first column is the
temperature at which at which a measurement was taken, T, and the second column is the
absorbance of the DNA at that temperature, A)
Create a Scatter Plot of A versus T (i.e., take A on the y
axis and T as the x-axis). Estimate Tm by drawing a
vertical line at a point half way between the lower portion
of the T-A curve (dsDNA) and the upper portion of the
curve (ssDNA). The value you determine is only an
approximation, especially if the upper and lower portions
of the curve is not flat.
Sample Melting Point Plot of DNA
0.9000
0.8000
0.7000
Absorbance
0.6000
0.5000
0.4000
0.3000
0.2000
0.1000
20
30
40
50
60
70
Temperature (oC)
80
90
100
Notice that the curve is sigmoidal (S-shaped). The melting point Tm is defined as the point
where the number of dsDNA base pairs is equal to the number of ssDNA base pairs. The
melting point corresponds to the midpoint of the steep increase in absorbance as
temperature increases.
Differentiating a Melting Point Data Set
Another method to determine the melting point is to differentiate the Absorbance (A)
with respect to Temperature (T). The maximum of this plot is another approximate value
for Tm.
Using calculus, the derivative of a function describes the slope (or change in slope) of that
function. Numerical differentiation of a data set to determine the slope of the function is
given by the following expression:
yn+1 - yn
Δy
Slope =
=
Δx
xn+1 - xn
Tm Using Excel
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The point at which there is a maximum change in the slope of the melting curve should
correspond to a maximum in the derivative of the curve. Therefore, numerically
differentiating a melting curve should provide a peak-shaped function with a peak
maximum at the melting point. This method is especially useful when the changes in
absorbance are small.
1. Calculate ΔT and ΔA from the
columns of the data set. This is done
by simply subtracting adjacent points,
so cell C3 contains the formula (+A3 A2) and cell D3 contains the formula
(+B3 - B2), etc.
2. Because you are taking the
difference between two adjacent
data points, you must calculate the
average temperature of the two
adjacent points. Create another
column of data so that cell E3
contains the formula +(A3 + A2)/2 and
cell E4 contains +(A4 + A3)/2, etc. This
can also be accomplished using the
AVERAGE function in Excel.
3. Calculate ΔA/ΔT. This is the slope or the numerical first derivative of the melting point
curve.
4. Plot ΔA/ΔT versus Tave. In this case, the x-axis values are E3:E16 and the y-axis values
are F3:F16. The resulting derivative plot is shown below. The melting point temperature is
the point where the slope of the derivative plot is located at the peak.
DNA Melting Point - Derivitave Plot
0.0040
0.0035
delta A / delta T
0.0030
0.0025
0.0020
0.0015
0.0010
0.0005
0.0000
20.0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
Tave
Tm Using Excel
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