Q.41
Four identical PCR reactions were carried out in tubes named I, II, III and IV.
Besides the usual mix of dNTPs, each of the tubes respectively contained
γ-32P dATP, β-32P dATP, α-32P dATP and
α-32P rNTP.
Which one of the tubes will have radiolabeled PCR product?
Options:
(A) Tube I
(B) Tube II
(C) Tube III
(D) Tube IV
Introduction
PCR labeling questions are common in molecular biology and biotechnology exams. To solve them correctly, one must understand how nucleotides are incorporated during DNA synthesis. In this article, we analyze which radioactive nucleotide will result in a radiolabeled PCR product and why the others fail.
Key Concept: How DNA Polymerase Uses dNTPs
A deoxynucleotide triphosphate (dNTP) has three phosphate groups:
α-phosphate − β-phosphate − γ-phosphate\alpha\text{-phosphate} \;-\; \beta\text{-phosphate} \;-\; \gamma\text{-phosphate}
During DNA synthesis:
-
Only the α-phosphate becomes part of the DNA backbone
-
β and γ phosphates are released as pyrophosphate (PPi)
👉 Therefore, only α-labeled nucleotides can label DNA
Tube-wise Analysis
Tube I: γ-³²P dATP
❌ Incorrect
-
γ-phosphate is released during polymerization
-
No radioactivity enters DNA
Tube II: β-³²P dATP
❌ Incorrect
-
β-phosphate is also released as PPi
-
No labeling of DNA
Tube III: α-³²P dATP
✅ Correct
-
α-phosphate is incorporated into DNA backbone
-
PCR product becomes radiolabeled
Tube IV: α-³²P rNTP
❌ Incorrect
-
PCR uses dNTPs, not rNTPs
-
DNA polymerase does not incorporate ribonucleotides
✅ Correct Answer
Option (C): Tube III
Summary Table
| Tube | Nucleotide Used | Incorporated into DNA? | Radiolabeled Product |
|---|---|---|---|
| I | γ-³²P dATP | ❌ No | ❌ |
| II | β-³²P dATP | ❌ No | ❌ |
| III | α-³²P dATP | ✅ Yes | ✅ |
| IV | α-³²P rNTP | ❌ No | ❌ |
Final Conclusion
Only α-³²P dATP results in a radiolabeled PCR product because DNA polymerase incorporates the α-phosphate into the growing DNA strand. Hence:
Correct Answer: Option (C) – Tube III
