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by Ivano Zara
Comments and observations are welcome. Please, write to ivano.zara.bio @ gmail.com

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DNA hybridization reactions
The content of these pages can be freely copied and distributed.
The only limitation: cite the author: Dr. Ivano Zara (ivano.zara.bio@gmail.com) web: dna_promix.it or biozarivan.it.
This work is still in progress. I wanted to publish the previews of the various chapters anyway, as I think they can be useful anyway.
To see this finished work, wait a bit. If you have any special requests, write to ivano.zara.bio@gmail.com

DNA hybridization reactions:
single strand A + single strand B ↔ double strand D
ssA + ssB ↔ dsD

The study of thermodynamics in DNA hybridization reactions is important to understand many of the applications (PCR, arrays, etc.) used in biology.
To design these reactions, the melting temperature is often used as a parameter.
Understanding what it is and how to estimate it is of fundamental importance for the success of the experiments.

I wanted to write these notes to describe in detail the thermodynamic reaction of hybridization of two single strands of DNA using, in particular, the nearest-neighbor method.

Differences in published data
I think that there is a lack of information on this topic, in fact, different sites offer heterogeneous estimation services that are inconsistent with each other.

In addition to adopting different methods and studies, some sites (even those of important companies, see in the next tab) make mistakes in applying the theory of DNA hybridization and use incorrect equations.
Other sites underestimate the importance of solutions and their salts.
To demonstrate some inconsistencies published on some sites, now, anticipate the equation to determine the melting temperature (which will be explored in more detail later) to report some published inconsistencies.

The general formula for determining the melting temperature should be:

Tm =
ΔH / ΔS R ln(Cx)
in °Kelvin (see explanation in the next chapters)

I will demonstrate that it is correct to replace Cx with the initial concentrations of reagents 'Ca' and 'Cb' in this way:
  • Generic case, always valid: Cx = Ca - ½ Cb;
  • In the case of PCR then Cx=Initial primer concentration.
  • In case the concentrations of the two reagents are equal Ca=Cb = C then Cx = ½ C


    • in PCR reaction:
    Some website use Cx= ½ [ primer concentration] (e.g. Promega).
    while others use Cx = ¼ [primer concentration] (e.g. Merck sigmaaldrich) ..
    Others use our annotation correctly Cx = [primer concentration] (e.g. Premier Biosoft).

    Link to our Oligo Melting application or PCR Primer Analysis.
These work
These notes are the result of a theoretical study of DNA hybridization chemistry and matured from the experience with Prof. Giorgio Valle's group at the Department of Biology of the University of Padua.
They are aimed at people (especially biologists) who already have the basis for understanding the structure of DNA, and who know the fundamentals of chemistry, in particular the basis of chemical reactions.

I have divided the work into several parts: