Polymerase Chain Reaction (PCR)

  • Thread starter _Mayday_
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Hey!

I have learned that one of the first steps in this technique involves heating the DNA sample to around 95 degrees. Now as far as I am aware the only thing holding the two Sugar Phosphate backbones together is the Hydrogen bonding between base pairs. Why is such a high temperature needed to break such weak bonds?

_Mayday_
 

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  • #2
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The DNA sample you're trying to amplify from could be a very long strand of DNA, so there could be a large number of hydrogen bonds that you'll have to break. You don't really want the strands to be associated with each other, so you use a relatively large amount of heat to help keep them from 'sticking' to each other.
 
  • #3
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Thank you Faustus.
 
  • #4
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I don't believe that temperature is really necessary, but just preferable because it can make the whole reaction go faster.

Originally it was not used at such temperature because the polymerase would denature at such high temperatures, then polymerases were adapted from thermophilic archaea, like Taq polymerase, which allowed the reaction to go at higher temps.
 
  • #5
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Not really. The actual polymerization temp is around 68-72° C depending on the polymerase. Some hot-start polymerase require activation at 94° C , though.
 
  • #6
Ygggdrasil
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The high temperature is needed to denature the DNA strands. Thermostable polymerases such as taq polymerase are required for the enzyme to survive the 95oC denaturation step. Otherwise, if one were to use regular E. coli DNA polymerase (as was done in the early days of PCR), one would have to cool to 37oC and add fresh enzyme during each cycle of the PCR reaction.
 

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