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Principle of the PCR - Polymerase Chain Reaction

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  • Principle of the PCR - Polymerase Chain Reaction

    I am lazy tonight.

    I made my best to create original Mingus'explanation in the lab most often, but tonight I had enough, sorry.

    Here is from
    http://users.ugent.be/~avierstr/principles/pcr.html

    Note that PCR is also a detection method, because we amplify a perticular and specific fragment of DNA in millions of copy enough to see it by our eyes via fluorescent dyes...

    The fragment amplified is always the one inside the position of our "primers" wich is a reactant in the the reaction mix.

    Each "target" have his pair of "primers".

    It is easy to develop them with the computational resources we have now.
    <center></center>
    <center>Principle of the PCR

    </center><hr> The purpose of a PCR (Polymerase Chain Reaction) is to make a huge number of copies of a gene. This is necessary to have enough starting template for sequencing.
    1. The cycling reactions : There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time.
      1. Denaturation at 94?C :
        During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for example : the extension from a previous cycle).
      2. Annealing at 54?C :
        The primers are jiggling around, caused by the Brownian motion. Ionic bonds are constantly formed and broken between the single stranded primer and the single stranded template. The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double stranded DNA (template and primer), the polymerase can attach and starts copying the template. Once there are a few bases built in, the ionic bond is so strong between the template and the primer, that it does not break anymore.
      3. extension at 72?C :
        This is the ideal working temperature for the polymerase. The primers, where there are a few bases built in, already have a stronger ionic attraction to the template than the forces breaking these attractions. Primers that are on positions with no exact match, get loose again (because of the higher temperature) and don't give an extension of the fragment.
        The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template)


      Figure 3 : The different steps in PCR. (pdf file of this picture)Animated picture of PCR Because both strands are copied during PCR, there is an exponential increase of the number of copies of the gene. Suppose there is only one copy of the wanted gene before the cycling starts, after one cycle, there will be 2 copies, after two cycles, there will be 4 copies, three cycles will result in 8 copies and so on.

      Figure 4 : The exponential amplification of the gene in PCR.
    2. Is there a gene copied during PCR and is it the right size ? Before the PCR product is used in further applications, it has to be checked if :
      1. There is a product formed.
        Though biochemistry is an exact science, not every PCR is successful. There is for example a possibility that the quality of the DNA is poor, that one of the primers doesn't fit, or that there is too much starting template
      2. The product is of the right size
        It is possible that there is a product, for example a band of 500 bases, but the expected gene should be 1800 bases long. In that case, one of the primers probably fits on a part of the gene closer to the other primer. It is also possible that both primers fit on a totally different gene.
      3. Only one band is formed.
        As in the description above, it is possible that the primers fit on the desired locations, and also on other locations. In that case, you can have different bands in one lane on a gel.


      Figure 5 : Verification of the PCR product on gel. The ladder is a mixture of fragments with known size to compare with the PCR fragments. Notice that the distance between the different fragments of the ladder is logarithmic. Lane 1 : PCR fragment is approximately 1850 bases long. Lane 2 and 4 : the fragments are approximately 800 bases long. Lane 3 : no product is formed, so the PCR failed. Lane 5 : multiple bands are formed because one of the primers fits on different places.

  • #2
    Re: Principle of the PCR - Polymerase Chain Reaction

    Animation
    http://www.dnai.org/b/index.html

    Found by goglicomplex, thanks

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    • #3
      Re: Principle of the PCR - Polymerase Chain Reaction



      A good thermocycler for PCR reaction of the 1st generation.

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      • #4
        Re: Principle of the PCR - Polymerase Chain Reaction



        Real-time PCR system of the 2nd generation.

        This time, no need to run agarose( a kind of tick gello) gel in elecrophorese(electricity in a solution) anymore.
        The machine read the reaction directly by a fluorescent probe.

        But we have to design this probe the same way we design reaction primers with a computer analysys work.

        It give us raw reading like that instead of a fluorescent band in a gel.

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        • #5
          Re: Principle of the PCR - Polymerase Chain Reaction



          The reactant, very small, very expensive.

          Around 200$ for 50ul ( microlitre ) of TAQ polymerase, a special polymerase that have the capability to work ar very high temperature (72oC) and to endure the work we expect from it ( around 38cycles ranging from 95oc to 60oC. )

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          • #6
            Re: Principle of the PCR - Polymerase Chain Reaction

            Reaction tube of around 20ul to 50ul of reaction mix.

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            • #7
              Re: Principle of the PCR - Polymerase Chain Reaction

              Putting reaction mix in the agarose gel


              Running the agarose gel

              Seeing the agarose gel on UV light with Dye

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