Melting temperature of the DNA and HRM

[lazyphant]

The melting temperature (Tm) of the DNA molecule is defined as the temperature at which half of the DNA strands are in the double-helical state and half are in the "random-coil" states.

I wonder why the Tm is defined like this. Why is not defined as temperature when both strands are completely separated. What is the benefit of the official definition.

Does the definition mean that the other end of the DNA-molecule is in double-helical state and other end in "random-coil" state? Or are there domains of double helix and domains of random coils (see the picture)?

I am about start use real-time-PCR machine and High Resolution Melting analysis function of it. The Tm of DNA is critical concept when analysing melting curves of DNA (at least I think so). That's why I want to understand what is the idea behind the official definition Tm of DNA.

(I posted this question also to the DNA forum, but I think this is better place for it)

[Aga]

Referring to my knowlegde I can say that the second of your two pictures is true.
Now why? Because the strands melt first in sequences where more A and T bases are present, and sequences with more clusters of G and C bases melt later. So when the half of the DNA strand is in the double-helical state and half is in the random-coil state it is more like there are domains of double helix and domains of random coils.

Now I don't exactly know why the definition of melting temp is formed as it is. I can only speculate that the definition is there to define some term so that everybody knows what they are referring to. Or maybe that the state of half denatured DNA is easier to observe than entirely melted and that's the reason? Or maybe the observation of 50% melted DNA compared with 70%, 80% or 100% melted DNA giver lower error of analysis?

[amicia]

In my opinion both the figures can represent what's happen during the melting. The first is more representative for short oligo like PCR products, while the latter for genomic DNA. To answer why the Tm is so defined I have the impression that is a simple definition, it's like the definition of infecting dose for the virus titolation (the dose in which 50% of cells or animal are infected) and there are many other definition that use the 50% as cut off value. Anyway this definition is very useful for the Real Time PCR cause when a melting resolution curve is done you can observe the logarithmic droop of fluorescence exactly when the 50% of DNA is melted.
Hope this opinion can help you. Which type of HMR system are you paining to buy?

[lazyphant]

Thanks for the replies,

I now have used RT-PCR three weeks. Now it is crystal clear how Tm is defined. It is the temperature where the denaturation of a particular DNA-molecule is the fastest. In other words Tm is only a mathematical parameter. See the picture in attachment:

Now I am wondering is the DNA concentration affecting the Tm values. If I have two identical DNA samples, which have different concentrations, will they give different Tm-values in melting analysis?

[amicia]

theorically no, but it's still possible. I suggest to carefully read this link:
[Only registered users see links. ]

[lazyphant]

Thanks for the post amicia!

Interesting. Although the shape of the melting curve should not be altered by the concentration, it will somehow change the curve shape when observed experimentally. Maybe Tm (or the melting curve shape) can be experimentally altered by concentration differences because Relative Fluorescence Units (RFU) are used to evaluate the amount of DNA. If there are different concentrations of the same sample (PCR-product) the melting curves will start from different level of initial fluorescence. Thus, the melting curve can be different? I am uncertain and confused.

[amicia]

To my best knowdelge many factors can influence the shape and Tm of the melting curve:
-the system you are using,
- the dye,
- the Mg concentration (could interfere with the dye too),
- the use of a passive reference dye control (ROX),
- the starting amount of DNA,
- the effiency of PCR.
For exemple I'm testing only on the basis of Tm (not on the basis of the shape curve) different bacterial strains using many dye and master mix. I found differences in the same sample of 3/5 °C on the basis of dye used.
I hope I've been of some help.

[lazyphant]

Originally Posted by amicia

To my best knowdelge many factors can influence the shape and Tm of the melting curve:
-the system you are using,
- the dye,
- the Mg concentration (could interfere with the dye too),
- the use of a passive reference dye control (ROX),
- the starting amount of DNA,
- the effiency of PCR.
For exemple I'm testing only on the basis of Tm (not on the basis of the shape curve) different bacterial strains using many dye and master mix. I found differences in the same sample of 3/5 °C on the basis of dye used.
I hope I've been of some help.

Thanks for the reply amicia!

If you can also explain the theory how the starting amount of DNA and the effiency of PCR affects shape and Tm of the melting curve, I would be very grateful.

-Lazyphant-

[lazyphant]

Originally Posted by amicia

To my best knowdelge many factors can influence the shape and Tm of the melting curve:
-the system you are using,
- the dye,
- the Mg concentration (could interfere with the dye too),
- the use of a passive reference dye control (ROX),
- the starting amount of DNA,
- the effiency of PCR.
For exemple I'm testing only on the basis of Tm (not on the basis of the shape curve) different bacterial strains using many dye and master mix. I found differences in the same sample of 3/5 °C on the basis of dye used.
I hope I've been of some help.

Thanks amicia!

If you could explain the theory how the starting amount of DNA and the effiency of PCR affects on the shape and the Tm of the melting curve, I would be very grateful.

Let me know your speculations, even if they are not perfect!

-Lazyphant-

[lazyphant]

Thanks amicia!

If you could explain the theory how the starting amount of DNA and the effiency of PCR affects on the shape and the Tm of the melting curve, I would be very grateful.

Let me know your speculations, even if they are not perfect!

-Lazyphant-