Question:
Give the steps in PCR or Polymerase chain reaction with a suitable diagram.
Answer:
(1) DNA segments and more than two primer molecules, four
Types of dNTP, thermostable DNA polymerases are mixed
together in an 'Eppendorf tube'.
 |
| Fig. Polymerase chain reaction |
(2) One PCR cycle is 3 - 4 min and includes
The following are the steps:
(a) Denaturation: The reaction mixture is heated at 90 - 98 °C.
This breaks the hydrogen bonds in DNA and two
DNA strands separate. This is called distortion.
(b) Annealing of primer: When the reaction mixture is cooled
At 40 – 60 °C, primers anneal with their complementary sequences
in ssDNA. This is called annealing.
(c) Extension of Primer: This step involves temp
increased to 70-75°C. At this temperature, thermostable
Taq DNA polymerase adds a nucleotide to the 3'end of the primer
Using single-stranded DNA as a template. This is called a primer
The duration of this expansion step is about two minutes.
(3) In an automatic thermal cycler, there are three steps above
Automatically repeats 20-30 times.
(4) Thus, at the end of the 'n' cycle there are 2n copies of DNA segments.
synthesized
