The mechanism of PCR

Question

Could anyone explain the The mechanism of PCR?

Answer 1

The polymerase chain reaction (PCR) is a technique widely used in molecular biology. It derives its name from one of its key components, a DNA polymerase used to amplify a piece of DNA by in vitro enzymatic replication. As PCR progresses, the DNA thus generated is itself used as a template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. With PCR it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. PCR can be extensively modified to perform a wide array of genetic manipulations.

Answer 2

watch this!

http://www.youtube.com/watch?v=_YgXcJ4n-...

I repeatidly am asked this question when talking about my job at http://www.mrgene.com . PCR is the key reaction in gene synthesis.

Answer 3

PCR is polymerase chain reaction, and is used to amplify (multiply) small amounts of DNA in order to work with a large sample of DNA. Before you begin PCR you need to understand a couple of things: the structure of DNA, base pairing (or complimentation), the effect of temperature on complimentation, the role of primers and DNA polymerase.

The process of PCR is cyclic. Which becomes important for automation (explained later)

Before anything, a fragment of DNA is isolated with the sequence of interest. The sequence before an after the sequence of interest must be known, in order to prepare primers, which will act as 'bookends' to amplification of the targeted sequence.

The Process:

1) The fragment of DNA is heated to 94-96C in order to denture (essentially split the two strand of DNA), creating two template strands.

2) Allowing the reaction to reach 50-65C to allow the annealing (gluing) of primers to the 5' end of each strand.

3) Heating the reaction to 75-80C to allow DNA polymerase to elongate a complimentary strand to the original template strands between the two primers. (dNTP's need to be provided: dATP, dCTP, dGTP,dTTP; these are the monomers of DNA)

4) Heating to 94-96C to allow the denaturing again (DNA strands split apart, DNA primers fall off, DNA polymerase falls off), and repeat the rest of the steps until a desired quantity of DNA is reached.

Things to note:

-There is an optimal time for each time we hold the reaction. Usually dependant on the length of DNA we are dealing with.

- The process of amplification is highly accurate, but decreases with accuracy as we use longer strands of DNA.

- This process takes hours, usually run in labsover night, so that in the morning your sample is ready.

- Can many very large amounts of DNA from very little amounts of extracted DNA (2 to the n'th power)

- Taq Polymerase, is the DNA polymerase used in PCR, this is not a human enzyme, but a bacterial one. This is special since it is from a bacterial that exists around thermal vents such as hot springs. The reason we use this polymerase is because its fuction is not changed by the high temperatures needed for the denaturing of DNA.

Automation:

Since the process is cyclic. Heating, Cooling, Heating, Cooling.... This process works beautifully automated by machinery. PCR machines are very common, and very little work (other than finding a DNA sample, and sequencing for primers) is needed by a technician.

Answer 4

PCR is also known as Polymerase Chain Reaction. It is basically a technique to amplify DNA. Mechanics: First you have a sample of the DNA you want to make more of. You would first denature this DNA into 2 single strands using high heat. Now the 2 single strands are floating. You then add the base pairs in a DNA strand (A,G,C,T) into the solution, and also add DNA Polymerase III. What DNA Polymerase 3 does is it binds corresponding base pairs to the DNA. For example, A to T, G to C.  So now, you let POL III do its work, and you got yourself 2 double helix DNA strands. Then you split those again, and repeat the process. This is usually done in a PCR machine.

Hope this helps.