DNA concentration determination

2 replies [Last post]
nicolep
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Joined: 07/19/2011

I have a question about your calcuations under hybridization of 2
different strands under the DINAMelt Web server. On this page you
can enter two oligo sequences.
Under the output page you can click on the text button underneath
the absorbance plot and get extinction coefficients for each
temperature. My question is: Is this the extinction coefficient for
measuring the molarity the "annealled DNA" made up of the two oligos
annealled together? I just wanted to be clear, because the graph
reads "oligo1sequence vs. oligo2sequence" and I wasn't quite sure
what that meant. I am trying to measure the concentration of 2
oligos with only partial complementarity. Thank you for your
consideration

venus666
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Joined: 07/05/2015
Hybridomas

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zukerm
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Joined: 11/12/2010
UV melting simulation

The plotted values are not "extinction coefficients". Extinction coefficients are published parameters that give UV absorbance for single-stranded RNA/DNA mono- or di-nucleotides. That is, they are the building blocks in computing the absorbance plots. The plotted values are absorbances at different temperatures and are weighted sums of extinction coefficients. The units are given along the "y" (vertical) axis in the plot. Sorry to be so pedantic.

In general, the UV absorbance plots tell you nothing about the molarity of the various molecular species at different temperatures. That is why we provide the concentration plots. Suppose that you simulate two oligos that are perfectly complementary. Suppose that the strand concentration of each oligo is 1μM and that full hybridization occurs at low temperatures. At low temperatures, there will be 1μM of the duplex in solution. If total melting occurs at high temperature, then there will be 1μM of the first strand and 1μM of the second, summing to 2μM. The concentration plots are always normalized so that the sum of the (up to) five plots is 1. They are "mole fraction" plots. The actual numbers are in a text file that can be downloaded.

Now let's go back to the absorbance plots. The assumption is that double-stranded RNA/DNA absorbs no light (UV). This is not true, but it gives a base line. As temperature increases, more and more nts become single-stranded and absorb light. For the case above, the plot should go from zero to a maximum value given by summing extinction coefficients for two single-stranded oligos. However, if hybridization is not perfect, or if folding occurs, creating a single-stranded hairpin loop, the base line is > 0 since even at the lowest temperatures, not all nts can be single-stranded. The plot simulates UV absorbance at 260 nM of the total "mess" in solution; which can be a complex combination of up to five molecular species. If you have a pair of oligos and you are pretty confident that they do not fold or self-hybridize, then the absorbance plots give you an accurate picture of the molarity of the hybrid at different temperatures. In general, the plot is the sum of absorbances for more than one molecular species, and there is no way to separate them by looking at the absorbance plot.

Does this help?