Question

In PCR...

A) Steps 1-3 are repeated 30-40 times. Why?

B) Is there a replication fork when you do replication in vitro like this? Explain.

Answer 1

PCR or Polymerase Chain Reaction is a technique which is used to produce millions of copies of the required DNA segment. The 3 steps involved in PCR include, Denaturation, Annealing and Extending. These three steps togetherly constitute a single cycle in PCR. These cycles are repeated 30-40 times because, as the number of cycles increases the amount of DNA synthesized increases as an exponent of two.(after first cycle, DNA content becomes 2¹ ie, 2, after second cycle, it becomes 2² ie, 4 after third cycle it becomes 2³ ie.,8 and so on..) So, as the prime aim of PCR is to increase the amount of DNS in a sample, the cycles are reapeated for about 30 to 40 times. Hence, by the end, DNA content ranges between 2³⁰ to 2⁴⁰ which is immensely a huge quantity.

When we do in vitro DNA synthesis, there is no formation of replication fork. In vivo conditions DNA has to melt (melting of hydrogen bonds) locally so, that DNA polymerase can use each of the single strands as template and can synthesisze DNA. This region where there occurs a local melting of hydrogen bond and a small bubble is formed is called as replication fork or replication bubble. But, when this process is carried out artificially (in vitro), during the denaturation step a temperature of about 94⁰c is used for the 2 strands to seperate completely by breaking of hydrogen bonds, and during the next step primers are annealed directly to the seperated strands. So, there doesnot occurs the formation of replication fork.