Which one of the following options represents a combination of terms that are matched INCORRECTLY?
(1) ddNTPs : Chain termination ddNTPs:
(2) South Western blot: Physical interaction between DNA and protein
(3) 5′ – 3′ exonuclease activity: Proof reading polymerase for PCR
(4) Yeast two hybrid system: Interaction between proteins

Incorrectly Matched Combination in Molecular Biology

In molecular biology, several techniques and terms are closely related to gene expression, protein-DNA interactions, and DNA sequencing. Understanding the accurate association of these terms is essential for experimental accuracy and effective research design. Let’s analyze the options to identify the incorrectly matched combination.

✅ Correct Answer: (3) 5′ – 3′ exonuclease activity: Proofreading polymerase for PCR

Analysis of the Given Options

(1) ddNTPs: Chain Termination – ✅ Correct

• ddNTPs (Dideoxynucleotide triphosphates) are modified nucleotides lacking a hydroxyl (-OH) group at the 3’ position.
• During DNA synthesis, when a ddNTP is incorporated into a growing DNA strand, it prevents further elongation because the absence of the 3′ OH group blocks the formation of a phosphodiester bond with the next nucleotide.
• This mechanism forms the basis of Sanger sequencing (chain termination sequencing).
  ✔️ Accurate match

(2) South Western Blot: Physical Interaction Between DNA and Protein – ✅ Correct

• South Western blotting combines Southern blotting (DNA detection) and Western blotting (protein detection).
• This technique is used to identify protein-DNA interactions by:
  • Separating proteins using SDS-PAGE
  • Transferring proteins onto a membrane
  • Probing the membrane with a radiolabeled DNA fragment
    ✔️ Accurate match

(3) 5′ – 3′ Exonuclease Activity: Proofreading Polymerase for PCR – ❌ Incorrect

• 5′ – 3′ exonuclease activity is NOT involved in proofreading during PCR.
• DNA polymerase I and other enzymes with 5′ – 3′ exonuclease activity are involved in:
  • Removal of RNA primers
  • Repair of damaged DNA strands
• Proofreading during PCR is carried out by 3′ – 5′ exonuclease activity (NOT 5′ – 3′).
• Taq polymerase, used in PCR, lacks proofreading activity, which is why high-fidelity polymerases like Pfu (with 3′ – 5′ exonuclease activity) are preferred for accurate DNA amplification.
  ❌ Incorrect match

(4) Yeast Two-Hybrid System: Interaction Between Proteins – ✅ Correct

• The yeast two-hybrid system is used to detect protein-protein interactions.
• In this technique:
  • One protein is fused to a DNA-binding domain (DBD).
  • The other protein is fused to an activation domain (AD).
  • If the two proteins interact, the transcription of a reporter gene (like LacZ) is activated.
    ✔️ Accurate match

Why 5′ – 3′ Exonuclease Activity is Incorrect for Proofreading

🔬 Exonuclease Activity and Proofreading

• 5′ – 3′ exonuclease activity – Removes RNA primers during lagging strand synthesis (DNA replication).
• 3′ – 5′ exonuclease activity – Removes mismatched nucleotides for proofreading.
• PCR polymerases like Pfu and Taq differ in proofreading ability:
  • Taq polymerase – No proofreading activity
  • Pfu polymerase – Has 3′ – 5′ proofreading exonuclease activity

Correct Function of 5′ – 3′ Exonuclease Activity

Summary of Correct and Incorrect Matches

Why Understanding Correct Matching Matters

1. Accurate Experimental Design:

   • Misunderstanding polymerase activity can lead to flawed PCR experiments.

2. Sequencing and Amplification:

   • Correct use of ddNTPs ensures successful chain termination and accurate sequencing.

3. Protein-DNA and Protein-Protein Interactions:

   • Techniques like South Western blot and yeast two-hybrid systems enable mapping of biomolecular interactions.

Common Enzymes Used in PCR and Sequencing

Conclusion

The incorrectly matched combination is (3) 5′ – 3′ Exonuclease Activity: Proofreading Polymerase for PCR. Proofreading polymerase activity is associated with 3′ – 5′ exonuclease activity, NOT 5′ – 3′ exonuclease activity. Understanding the correct functions of exonucleases, ddNTPs, and molecular biology techniques is essential for successful experimental outcomes.

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