PCR technique and DNA denaturation

Which of the following statements about PCR is incorrect?
a. The DNA template is denatured using helicase.
b. PCR is used to obtain millions of copies of a specific region of DNA.
c. A thermostable DNA polymerase is used because of the high temperatures required in PCR.
d. Primers are needed because DNA polymerase cannot initiate synthesis, but can only elongate from an existing 3’OH.

Correct Answer: ✅ (a) The DNA template is denatured using helicase.

Explanation

The Polymerase Chain Reaction (PCR) is a widely used molecular biology technique that allows the amplification of a specific DNA sequence through a series of controlled temperature changes. The key steps involved in PCR include:

🔬 1. Denaturation

• In PCR, the DNA double strands are separated (denatured) by heating to 94–98°C.
• No helicase enzyme is involved — the high temperature alone is sufficient to break the hydrogen bonds between the complementary DNA strands.

🧬 2. Annealing

• Primers (short DNA sequences) bind to the complementary sequences on the single-stranded DNA.
• Annealing typically occurs at 50–65°C depending on primer length and GC content.

🏆 3. Extension

• A thermostable DNA polymerase (e.g., Taq polymerase from Thermus aquaticus) synthesizes a new strand by adding nucleotides to the 3’ end of the primer at 72°C.

✅ 2. Why the Other Statements Are True

🔥 3. Why Helicase is NOT Used in PCR

• In living cells, helicase unwinds DNA during replication by breaking hydrogen bonds between base pairs.
• In PCR, high heat (94–98°C) replaces the need for helicase by breaking the hydrogen bonds and separating the DNA strands.
• Helicase would not survive at such high temperatures because it is not heat-stable like Taq polymerase.

🧪 4. Key Components of PCR

5. Steps of PCR

Step 1: Denaturation

• Temperature: 94–98°C
• Breaks hydrogen bonds → Separates DNA strands

Step 2: Annealing

• Temperature: 50–65°C
• Primers bind to complementary DNA sequences

Step 3: Extension

• Temperature: 72°C
• Taq polymerase synthesizes new DNA strand

🌡️ 6. Why Taq Polymerase is Used

✅ Taq polymerase is isolated from the thermophilic bacterium Thermus aquaticus → Stable at high temperatures.
✅ It withstands repeated cycles of heating and cooling without denaturation.

7. Applications of PCR

1. Medical Diagnostics

• Detecting pathogens (e.g., COVID-19, HIV)
• Genetic disorder screening

2. Forensic Science

• DNA fingerprinting
• Crime scene analysis

3. Evolutionary Biology

• Studying genetic variation
• Analyzing ancient DNA samples

4. Research and Biotechnology

• Cloning and gene editing
• Mutation analysis

Summary

• PCR is a powerful technique for amplifying DNA.
• DNA is denatured by heat, not helicase.
• Taq polymerase is used for its thermostability.
• ✅ Correct Answer: (a) The DNA template is denatured using helicase.