Question

Why do scientists do experiments using PCR? More specifically, what are the temperatures used for PCR and what are the objectives for each temperature?

Answer 1

PCR ( polymerase chain reaction) is a in-vitro method for amplification or generating multiple copies of DNA segment.It is developed by Kary Mullis .scientists use it as a alternative to normal gene cloning in which a vector and host is required for cloning. Scientist use PCR for experiment than normal gene cloning because of its advantages. It it has very high sensitivity so can detect even a single copy of target DNA in sample provided and generate millions of copies of it. Second advantage of PCR is that it require very low amount of DNA in nanograms in contrast to gene cloning which require in micrograms. PCR procedure is very fast or rapid require only hours, one the other hand gene clonning require days or even weeks. It is also preferred because it doesnot require costly DNA restriction enzymes, DNA ligase and vector DNA which required in normal gene clonning experiment. Because of all these reason scientists use PCR for there experiment.

For setting a PCR eeaction following components are required:

a)Template DNA: also sometimes known as target DNA.

b) Two oligonucleotide primers: Two primers of around 20 basepair long. They are complementary to two 3' end of template DNA.

c) deoxynucleotide phosphatidates( dNTPs) : four dNTPs i.e dATP, dGTP, dCTP and dTTP for forming new DNA copies

d) enzyme : DNA polymerase enzyme is used for this process. Commonly Taq polymerase is used derived from Thermus aquaricus.

There are three stages in PCR: Denatiration, annealing and extension.All three have different temperatures

1)Denaturation : In this stage reaction mixture is provided with temprature between 90 to 98 degree calcius. Community used temprature is 94 degree calcius. This temprature is provided for denaturation of DNA so that both strands seperate from each other. Each single strand than act as template for DNA synthesis. Duration of this step is around 2 minutes.

2) Annealing: During the annealing step temprature is reduced to between 40 to 60 degree calcius. This is to permit annealing of primers to complementary sequence of template DNA. The duration of this step is 1 minute. This temprature is annealing temperature of our primers.

3) Primer extension: In this step temprature is adjusted so that DNA polymerase synthesis complimentary strand using 3' OH end of primer for extension. The extension is like synthesis of leading strand of DNA replication. In this stage temprature is around 72 degree calcius for 2 min. This is optimum temprature for DNA polymerase to synthesis DNA or for extension.

After completion of first cycle, second cycle begins repeating steps of Denaturation, annealing and extension. At the end a final extension is also performed when all cycles set for PCR completes.

Steps are also shown diagrammatically in below image