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What is the principle of real time PCR?

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Question added by zainab zainii , principle investigator , university of the Punjab Lahore
Date Posted: 2014/03/02
rakan naboulsi
by rakan naboulsi , Researcher , Uppsala University

PCR stands for Polymerase Chain Reaction. Its principle is easy. You add dNTP (De-oxy-Nucleotide Tri-Phosphate) that are the building blocks of DNA; you also add a polymerase (the enzyme that will synthesize DNA using dNTP) and a buffer that supports the function of the polymerase. Then you add forward and reverse primers (a short sequence of DNA that will anneal to its complementary sequence in the DNA sample). Finally you add the DNA sample.

The primers will anneal to their complementary sequence in the DNA at a specific temperature. The enzyme binds to the double stranded DNA-primer and will start synthesizing the DNA fragment between the two primers.

 

PCR is an efficient reaction that will lead to double the amount of DNA with every cycle. Supposing that the efficiency was100% and you had only one DNA fragment in you sample; after the first cycle, there will be two. Then after the second cycle there will be4. Then it continues as8,16,32,64...

 

After30 to40 cycle there will be a very high number of the amplified DNA that will be enough to proceed with further experiments.

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