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Thread: DNA hybridisation (investigation to establish evolutionary relationships between organisms)

  1. #1 DNA hybridisation (investigation to establish evolutionary relationships between organisms) 
    j.r
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    When DNA hybridisation experiments are carried out, why do the experimenters measure the temperature at which half the DNA has split into its two strands? I have heard that they measure the temperature when half the sample splits into the two strands it's made from. But I don't really understand why they record the temperature at half rather than when the strand has completely spit into two? I'd be very grateful if anyone may be able to help me understand a little better. Thank you very much!


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    Forum Professor Zwirko's Avatar
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    Technically it's not really a melting point as such, even though it is referred to as a melting point. Think of it as the midpoint of a phase transition rather than a traditional melting point temperature. So, "Tm" would be the temperature midpoint at which the equilibrium state consists of equal amounts of double stranded and single stranded molecules and the standard free energy is zero. The midpoint is related to the stability of the molecule and therefore its composition.

    As to why the midpoint temperature us used, I'm not really sure. It might be a convenient reference point, make calculations easier, make measurement easier or reflect some deeper thermodynamic principles. Or maybe none of the above...?


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    HFS
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    Each strand of DNA is bound to the other by hydrogen bonds between the nucleotide bases. The adenine-thymine pair is bound by two hydrogen bonds, and the guanine-cytosine pair is bound by three hydrogen bonds. DNA double-helices have varying GC:AT content; greater GC content = more hydrogen bonds = higher Tm. Bacteria are often categorised in this way - high GC and low GC content. More information on this wiki page: GC-content - Wikipedia, the free encyclopedia
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    Quote Originally Posted by HFS View Post
    Each strand of DNA is bound to the other by hydrogen bonds between the nucleotide bases. The adenine-thymine pair is bound by two hydrogen bonds, and the guanine-cytosine pair is bound by three hydrogen bonds. DNA double-helices have varying GC:AT content; greater GC content = more hydrogen bonds = higher Tm. Bacteria are often categorised in this way - high GC and low GC content. More information on this wiki page: GC-content - Wikipedia, the free encyclopedia
    Well yes, but i think this is to much trouble for simply checking GC content, as there are way easier ways to do it. Simply use 30 primers in a coctail, and see which ones the DNA uses. (labelled ofcourse)

    Though this experiment may calculate the exact energy requirement. Simply gradually imput energy, and measure temperature in an enclosed system. The moment where the line halts, the energy will be solely used to wring the DNA off eachother. Morely based on the 3 dimensional structure, as well as some of the proteins there can be still on the DNA.

    As the more curled up, full of proteins, and more GC over AT connections there (also some epigenetics involved there) the more energy required.

    They really still do this for simple GC content? Isn't a simple sequence analyser a lot more precise?
    Growing up, i marveled at star-trek's science, and ignored the perfect society. Now, i try to ignore their science, and marvel at the society.

    Imagine, being able to create matter out of thin air, and not coming up with using drones for boarding hostile ships. Or using drones to defend your own ship. Heck, using drones to block energy attacks, counterattack or for surveillance. Unless, of course, they are nano-machines in your blood, which is a billion times more complex..
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    HFS
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    Hmm, I disagree that it's much trouble, all it requires is a DNA extraction and a thermal-controlled spectrophotometer assay, it would be done before you even had a PCR set up. Of course if you had the whole genome sequence then it's more precise, but the spectrophotometer method has been used pre-genomic era and there's a wealth of data on the GC content of non-sequenced species. So, pros for the method I outlined: 1. cheaper, 2. quicker, 3. more comparable data available. Pros for yours: 1. more accurate. I know which one I'd choose. No-one I know genome sequences just to see GC content.
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