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RAPD - close proximity of primers

  1. Oct 29, 2018 #1
    I was reading RAPD kit manual by himedia and couldn't understand why the two primers should be at close proximity and in opposite orientation to actually amplify DNA.

    'In RAPD analysis, for PCR to occur: a) the primers must anneal in a particular orientation (such that they point towards each other) and, b) they must anneal within a reasonable distance of one another. Successful primer pairs produce different banding profiles of PCR products between individuals, strains, or species when analyzed by gel electrophoresis.'
     
    Last edited: Oct 29, 2018
  2. jcsd
  3. Oct 29, 2018 #2

    BillTre

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    The polymerase enzyme often used on PCR (Taq polymerase), although good at making short strands of DNA, does not generate long strands that well.
    This limits most PCR amplification to shorter strands.
    Thus the primers work better when close together.
     
  4. Nov 6, 2018 #3
    Thank you so much.
     
  5. Nov 14, 2018 #4
    But BillTre, won't the Taq polymerase extend any primer irrespective of its orientation. All it needs are a suitable buffer, ions, temperature and dNTPs. Why should proximity be an issue when the primers are binding to two completely different strands?
     
  6. Nov 14, 2018 #5

    BillTre

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    It will extend from any primer, however the amplification requires that a primer bind to the product of a previous extention (after the process has been initiated on the first round from the starting DNA) and run back, extending the opposite strand to produce a double stranded product that will have primer binding sties on opposing strands.
    Both of the strands from these will new short double stranded products will be able to bind primers and produce new product in the next round.
    In this way, the number of products grow exponentially as new rounds of PCR reaction occur.
    Other wise the growth of products will only be linear: a copy is made, things are denatured, new primers are bound and a new copy is made only on the starting material, not on the newer amplified strands since the binding site (the opposite strand) for the original primer has not been reproduced in the new material.

    Wikipedia has a decent picture showing how this works.
     
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