Question

Which of the following activity is not present in Taq polymerase?

• A. 5'–3' polymerase
• B. 5'–3' exonuclease
• C. 3'–5' exonuclease
• D. Both 5'–3' polymerase and 5'–3' exonuclease

Hint:

Taq polymerase lacks proofreading ability because it does not have 3'–5' exonuclease activity, making it prone to errors during amplification.

Answer 1

The Correct Option is C

Step 1: Understanding Taq polymerase.
Taq polymerase is a thermostable DNA polymerase used in the polymerase chain reaction (PCR) to amplify DNA. It has several key activities: 5'–3' polymerase activity, which allows it to synthesize DNA, and 5'–3' exonuclease activity, which allows it to degrade DNA. However, Taq polymerase lacks 3'–5' exonuclease activity, which is present in some other polymerases for proofreading.
Step 2: Analyzing the options.
(1) 5'–3' polymerase: This is present in Taq polymerase. It is responsible for adding nucleotides during DNA synthesis.
(2) 5'–3' exonuclease: This is present in Taq polymerase. It allows for the degradation of DNA.
(3) 3'–5' exonuclease: Correct — Taq polymerase lacks 3'–5' exonuclease activity, which is responsible for proofreading during DNA synthesis.
(4) Both 5'–3' polymerase and 5'–3' exonuclease: This is incorrect. Both of these activities are present in Taq polymerase.
Step 3: Conclusion.
The correct answer is (3) 3'–5' exonuclease, as Taq polymerase does not have proofreading activity.

Answer 2

Instead of listing Taq's activities from memory, we can reason from what PCR needs and what problems arise in practice, then check each option against that.

1. 5'-3' polymerase: PCR fundamentally requires an enzyme that extends a primer by adding nucleotides in the 5' to 3' direction using the template strand; Taq performs this efficiently and is thermostable enough to survive repeated denaturation cycles, so this activity is present.
2. 5'-3' exonuclease: This activity is exploited deliberately in TaqMan probe-based real-time PCR, where Taq cleaves a fluorescent probe annealed downstream as it extends the primer. Since this application only works because Taq has this activity, it must be present.
3. 3'-5' exonuclease: This is the proofreading function that removes a wrongly incorporated nucleotide from the growing 3' end. A well-known practical consequence of PCR using Taq is a comparatively higher error rate than proofreading enzymes like Pfu -- a direct sign that this proofreading activity is missing from Taq.
4. Both 5'-3' polymerase and 5'-3' exonuclease: Since both of these activities are individually confirmed present, an option that groups them together as "not present" cannot be correct.

The higher mutation rate observed with Taq-based PCR, compared with proofreading polymerases, is explained precisely by its lack of 3'-5' exonuclease activity.

Therefore, the correct answer is 3'-5' exonuclease.