8.4.4 Polymerase Chain Reaction

Steps in PCR

1. Setting up the Reaction Mixture:

• Combine the following components:
• DNA sample (the target DNA to be amplified).
• Primers (short sequences of nucleotides that bind to the DNA at specific points).
• Free nucleotides (dNTPs: A, T, C, G).
• DNA polymerase (enzyme that synthesises the new DNA strands, e.g., Taq polymerase).

2. Denaturation (95°C):

• The mixture is heated to 95°C, breaking the hydrogen bonds between DNA strands.
• This separates the DNA into two single strands.

3. Annealing (50–65°C):

• The temperature is lowered to 50–65°C (depending on the primers).
• Primers bind (anneal) to their complementary sequences on the single-stranded DNA.

4. Extension (70°C):

• The temperature is raised to 70°C, the optimum for Taq polymerase (a heat-stable enzyme from bacteria found in hot springs).
• Taq polymerase adds free nucleotides to the primers, synthesising a new complementary DNA strand.

5. Repeat Cycle:

• These three steps (denaturation, annealing, extension) are repeated ~30 times.
• With each cycle, the amount of DNA doubles, producing millions of copies in a few hours.

Key Features of PCR

• Exponential Amplification: Each cycle doubles the DNA, leading to exponential growth.
• Specificity: The use of primers ensures only the target DNA sequence is amplified.
• Speed: A full cycle takes just a few minutes, allowing millions of copies to be created quickly.

Applications of PCR

• Forensics: Amplifying DNA from crime scenes for identification.
• Medical diagnostics: Detecting genetic mutations, infections, or diseases (e.g., COVID-19 testing).
• Genetic research: Cloning, sequencing, and analysing specific DNA regions.