The region of DNA shown below is to be amplified by PCR. The appropriate pair of primers to use is which of the following?
(A) B and C
(B) A and D
(C) A and C
(D) B and D

Correct Answer: ✅ (A) B and C

Explanation

PCR (Polymerase Chain Reaction) is a widely used technique to amplify a specific region of DNA. Successful PCR requires a pair of primers — a forward primer and a reverse primer — that bind to opposite strands of the target DNA in a specific orientation.

🔬 1. How PCR Primers Work

• Forward Primer: Binds to the 5′ end of the sense strand (template strand).
• Reverse Primer: Binds to the 3′ end of the antisense strand (complementary strand).
• DNA polymerase extends the primers in the 5′ → 3′ direction, producing new DNA strands.

✅ Why the Answer is (A) B and C

1. Primer B – Acts as the forward primer, binding to the 5′ end of the template strand.
2. Primer C – Acts as the reverse primer, binding to the 3′ end of the complementary strand.
3. Both primers face each other, allowing DNA polymerase to extend them and produce the desired amplified product.

❌ Why Other Options Are Incorrect

How to Choose the Right PCR Primers

✅ 1. Correct Orientation:

• Forward primer binds to the 5′ end of the template strand.
• Reverse primer binds to the 3′ end of the complementary strand.

✅ 2. Proper Length:

• Ideal primer length: 18–24 nucleotides
• Short primers → Low specificity
• Long primers → Low efficiency

✅ 3. Melting Temperature (Tm):

• Ideal Tm: 50°C–60°C
• Tm can be calculated using the formula:

$$\text{Tm}=(4\times G+C)+(2\times A+T)$$

✅ 4. GC Content:

• Ideal GC content: 40%–60%
• High GC content → Stronger binding and higher stability

✅ 5. Avoid Primer-Dimer Formation:

• No complementary regions within the primers
• No secondary structure formation

Steps in PCR Amplification

🧪 1. Denaturation:

• Heating to 94–98°C → Breaks hydrogen bonds → Single-stranded DNA templates

🧬 2. Annealing:

• Cooling to 50–65°C → Primers bind to target DNA sequences

🔎 3. Extension:

• DNA polymerase extends the primers at 72°C → Formation of new DNA strands

Cycle-Wise DNA Amplification

Common Errors in PCR Primer Design

🔴 1. Wrong Orientation:

• Primers should face each other → Incorrect orientation = No amplification

🔴 2. Primer-Dimer Formation:

• Complementary sequences in primers → Leads to unwanted products

🔴 3. Incorrect Tm:

• Low Tm → Non-specific binding
• High Tm → Weak primer binding

Applications of PCR

✅ 1. Genetic Diagnosis:

• Detecting genetic mutations and infections

✅ 2. Cloning and Sequencing:

• Amplifying genes for functional analysis

✅ 3. Forensic Science:

• DNA fingerprinting and criminal identification

✅ 4. Evolutionary Studies:

• Amplification of ancient DNA for phylogenetic analysis

Summary

• PCR primers must bind in opposite orientations on complementary DNA strands.
• Primer B binds to the 5′ end of the template strand, and Primer C binds to the 3′ end of the complementary strand.
• This ensures correct amplification of the target region.
• ✅ Correct Answer: (A) B and C