Nucleic acid labeling methods: which radioactive nucleotide is used for 5′ end-labeling of oligonucleotides? Select the reagent typically employed with T4 polynucleotide kinase for end-labeling.

Introduction / Context:Radiolabeling oligonucleotides is a classic technique for sensitive detection in hybridization assays and kinase reactions. Different isotopic positions serve distinct purposes: internal labeling for synthesis versus end-labeling for probe tagging.

Given Data / Assumptions:

• End-labeling targets the 5′ terminus of an oligonucleotide.
• T4 polynucleotide kinase transfers a phosphate to the 5′-OH group.
• Choice of nucleotide determines where the radioisotope ends up.

Concept / Approach:T4 polynucleotide kinase transfers the terminal gamma-phosphate from ATP to the 5′ end of DNA or RNA. Therefore, gamma-32P-ATP is required. Alpha-32P-nucleotides are used for polymerase-mediated internal labeling, not kinase end-labeling.

Step-by-Step Solution:Identify the enzyme: T4 polynucleotide kinase.Identify the phosphate donor position: the gamma-phosphate of ATP.Select gamma-32P-ATP to ensure the isotope is transferred to the 5′ end.

Verification / Alternative check:Autoradiography after PAGE shows strong terminal signals when gamma-32P is used; alpha-32P incorporation does not occur in kinase-only reactions.

Why Other Options Are Wrong:Alpha-32P nucleotides label internally during polymerization. “Any of these” is incorrect because position matters. Tritium-labeled thymidine is used in cell proliferation assays, not in 5′ end-labeling with kinase.

Common Pitfalls:Confusing nick translation or random priming (alpha-32P) with kinase end-labeling (gamma-32P).

Final Answer:(gamma)-32P-ATP.