Question

Describe the steps involved in a Polymerase Chain Reaction (PCR) technique.

Hint:

Using a thermostable enzyme like Taq polymerase is crucial: standard DNA polymerases would denature and lose all activity during the high-temperature denaturation step ($95^\circ\text{C}$).

Answer 1

Step 1: Definition and Purpose
The Polymerase Chain Reaction (PCR), developed by Kary Mullis in 1985, is an in vitro technique used to exponentially amplify a specific target DNA segment.

Step 2: The Three Steps of a PCR Cycle
Each cycle consists of three sequential, temperature-controlled steps performed in a thermal cycler:
1. Denaturation ($94^\circ\text{C} - 96^\circ\text{C}$ for $30-60$ seconds):
- The reaction mixture is heated to a high temperature.
- This breaks the hydrogen bonds between complementary bases, separating the double-stranded DNA template into two single strands.
2. Primer Annealing ($50^\circ\text{C} - 65^\circ\text{C}$ for $30-60$ seconds):
- The temperature is lowered to allow two synthetic oligonucleotide primers to bind to their complementary sequences at the 3'-ends of the single-stranded DNA templates.
3. Primer Extension ($72^\circ\text{C}$):
- The temperature is raised to the optimum for Taq DNA Polymerase (a thermostable enzyme isolated from the hot spring bacterium Thermus aquaticus).
- The enzyme adds dNTPs to the 3'-OH end of the primers, synthesizing a new complementary strand in the 5' to 3' direction.


Step 3: Exponential Amplification
Repeating this cycle 25 to 35 times results in the exponential amplification of the target sequence. Final Answer: The three steps of PCR are (1) Denaturation at $95^\circ\text{C}$ to separate the double-stranded template, (2) Annealing at $55^\circ\text{C}$ to hybridize primers to the single strands, and (3) Extension at $72^\circ\text{C}$ using Taq polymerase to synthesize the new complementary strands.