Question
Give the steps in PCR or polymerase chain reaction with suitable diagrams.
✓
Answer
(1) The DNA segment and excess of two primer molecules, four types of dNTPs, the thermostable DNA polymerase are mixed together in ‘eppendorf tube’.
(2) One PCR cycle is of 3-4 minutes duration and it involves following steps:
- Denaturation : The reaction mixture is heated at 90-98°C. Due to this hydrogen bonds in the DNA break and two strands of DNA separate. This is called denaturation.
- Annealing of primer : When the reaction mixture is cooled to 40-60°C, the primer pairs with its complementary sequences in ssDNA. This is called annealing.
- Extension of primer : In this step, the temperature is increased to 70-75°C. At this temperature thermostable Taq DNA polymerase adds nucleotides to 3’end of primer using single-stranded DNA as template. This is called primer extension. Duration of this step is about two minutes.
(3) In an automatic thermal cycler, the above three steps are automatically repeated 20-30 times.
(4) Thus, at the end of ‘n’ cycles 2n copies of DNA segments, get synthesized.
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