Please refer to the attached Figure, which has three parts, A,B and C.
I first annealed complementary ssDNA in a PCR machine. Then, I heated the annealed dsDNA from 25 deg to 75 deg until all the DNA melts (Fig A). The DNA duplex has a melting temp (Tm) of around 50 deg. The experiment was done using the UV spectrometer, where the annealed DNA was put in a cuvette attached to a temperature controller (Fig B). Fig C shows a plot of Absorbance vs Temp. Data was collected for temperatures from 25 to 75 deg, at an interval of 5 deg.
As you can see in Fig C, the absorbance changes from a minimum of 0.5 to a maximum of 0.7. At 25 deg, I would expect the sample to completely be composed of dsDNA, whereas at 75 deg, I expect only ssDNA (since my melting temperature is around 50 deg). Thus, shouldn't my absorbance double from 0.5 to 1, instead of going from 0.5 to 0.7? The absorbance curve seems to be a bit weird. Typically, the change in absorbance around the Tm is not this slow, but very rapid. And at high temperatures, it should completely saturate. In my data, it does not saturate. Does this indicate that my DNA has not completely melted?
You expect that having twice as many molecules in solution (100% vs. 0% melted) will lead to a doubling of absorbance, but that is based on the assumption that the molar absorbtivities (i.e. molar extinction coefficients) of ss and dsDNA are the same. Is that the case? The "Quick Conversions" section of the page linked below suggests that 50 µg dsDNA and 37 µg ssDNA have the same absorbance, suggesting that ssDNA has an absorbtivity that is ~1.35X that of dsDNA. You have the same mass of DNA throughout, but convert completely from dsDNA to ssDNA, so you would expect your absorbance to increase from 0.5 to (0.5 x 1.35) = 0.68 -- which is close to what you see.
https://www.sigmaaldrich.com/technical-documents/articles/biology/quantitation-of-oligos.html
Albert Vill , thanks for the information, and the link. While that does explain why my absorbtivity doesn't double, the shape of the curve itself is a bit strange. If my dsDNA does indeed melt completely to ssDNA, my absorbance at high T should saturate towards the end. However, the absorbance continues to increase, as you can see in Fig C. Is that supposed to happen? In addition, do you feel that the change in absorbance around the Tm should be more rapid than what my data shows?
Ranjit Gulvady -- the rate of denaturation around your Tm is going to depend on your DNA sequence and buffering conditions. It's hard to know whether that is a feature of your specific experiment or just an anomaly without more replicates. As for why the absorbance doesn't saturate, I'm not sure. But you also see a gradual increase in absorbance for points below your Tm, which may indicate there is some temperature-sensitive component of your system outside of the DNA itself.
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