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PCR and Taq polymerase

  1. Jul 9, 2015 #1

    Suraj M

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    I recently learnt a bit about PCR,
    they say that we heat DNA to denature it, upto 90°C and then we cool it down to 72°C and add Taq polymerase( though we use Pfu nowadays)
    My question is, when you add Taq pol. then it replicates DNA, does it result in the formation dsDNA or ssDNA?
    From what I've seen ( in text books and diagrams) they say it is dsDNA but at 72°C can dsDNA exist without getting denatured?(<70°C required for A/T bond)
     
  2. jcsd
  3. Jul 9, 2015 #2
    The polymerase reads one strand and makes a single strand at a time, however somewhere else in the tube, the antisense strand is also being made by another polymerase, later when the temperature is dropped those two homologous strands unite, and effective a double stranded DNA has been made. So while each polymerase is only make a single strand at a time, effectively dsDNA is being made.
     
  4. Jul 9, 2015 #3

    Suraj M

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    the daughter need not pair with any particular antisense strand after cooling right?
    so when Taq pol. acts only ssDNA is formed, could you please provide a reference, i needed it for something, please.
    Thank you for replying
     
  5. Jul 10, 2015 #4
    dsDNA is already 'finished'.

    Only after denaturing, strands become single. After a primer fuses(it can because it is short and temperature/thermodynamics now favour hydrogen bonding) with the strand, the taq can then create a complement strand to the ssDNAs to make them dsDNAs. There will be hydrogen bonds between the two strands, the template and the new, and at 72 degrees the temp will be too low for denaturing. The new strand and the template will stay together, just as in a cell.

    Complete strands are so long, they cannot find the conformation that is most thermodynamically favourable, like a protein can, because it has to in the exact perfect conformation, where every basepair aligns with the complementary base pair, all 100s to 1000s of them.

    For proteins there is the Levinthal's paradox, but there it is a paradox as there is a solution namely many intermediates that funnel towards the final conformation. For DNA, no such state exists. Once big DNA molecules are denatured, they won't be able to return to the minimum energy conformation. Primers are short enough so that they can find the correct conformation.

    The inside of a DNA strand is hydrophobic; it does not want to interact/exposed to water. The base pairs with their pyrimidines and purines are hydrophobic. If you put a ssDNA in water, it will try to minimize base pair interactions with water, but it cannot by chance line up with it's complementary strand, were you to also put it in that solution as a ssDNA.

    Of course changing the temperature changes if entropy or enthalpy wins out. At high temperatures, randomly mixing becomes more dominant. At lower temperatures, lower energy/favorable interactions, but more ordered, structures dominate.

    https://upload.wikimedia.org/wikipe...n.svg/840px-Polymerase_chain_reaction.svg.png
     
  6. Jul 10, 2015 #5

    Suraj M

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    So annealing happens only at 68°C?
    So then at 72°C if i add DNA to the substrate then it wouldn't get denatured? and the other denatured ones will stay denatured? Is that what you are implying?
     
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