How can use lable oligo nucleotide (GGATATCC) with any reagents
P. [Y-32P] ATP
Q. polynucleotide kinase
R. DNA-dependent RNA polymerase
S. DNA ligase

A. P and Q
B. R and S
C. P and r
D. Q and S

How to Label Oligonucleotides Using Polynucleotide Kinase and γ-32P ATP

Oligonucleotide labeling is a crucial step in molecular biology experiments, especially for studying DNA-protein interactions, nucleic acid hybridization, and Southern or Northern blotting. The most commonly used method for labeling oligonucleotides involves using polynucleotide kinase (PNK) and γ-32P ATP. This method ensures high sensitivity and specificity, making it ideal for a wide range of molecular biology applications.

Correct Answer:

The correct answer is A. P and Q
👉 γ-32P ATP (P) and polynucleotide kinase (Q) are used for labeling oligonucleotides.

Understanding Oligonucleotide Labeling

Oligonucleotides are short, single-stranded DNA or RNA molecules that can be labeled using various radioactive or fluorescent markers. Labeling enhances the detection and analysis of these molecules in various biochemical and molecular biology assays.

The most effective method for labeling oligonucleotides is 5′-end labeling using polynucleotide kinase and γ-32P ATP.

How Polynucleotide Kinase and γ-32P ATP Work

1. Polynucleotide Kinase (PNK)

• Polynucleotide kinase is an enzyme that transfers a phosphate group from ATP to the 5′ hydroxyl (5′-OH) of oligonucleotides.
• The enzyme requires magnesium (Mg²⁺) ions for activity.

2. γ-32P ATP

• γ-32P ATP is a radiolabeled ATP molecule.
• The radioactive phosphorus-32 (32P) is located at the gamma position of ATP.
• When PNK transfers the gamma phosphate to the 5′-OH of the oligonucleotide, the resulting labeled oligonucleotide can be detected using autoradiography or phosphorimaging.

Step-by-Step Procedure for Oligonucleotide Labeling

Step 1: Reaction Preparation

 Prepare the reaction mixture:

• Oligonucleotide (with free 5′ hydroxyl group)
• γ-32P ATP (high specific activity)
• T4 Polynucleotide Kinase (PNK)
• Buffer containing Mg²⁺ ions

Step 2: Incubation

Incubate the mixture at 37°C for 30 minutes.

• PNK transfers the gamma-labeled phosphate from γ-32P ATP to the 5′-OH of the oligonucleotide.

Step 3: Termination

Stop the reaction by adding EDTA (to chelate Mg²⁺).

Step 4: Purification

 Purify the labeled oligonucleotide using a desalting column or gel filtration to remove unincorporated γ-32P ATP.

Step 5: Detection

 Analyze the labeled oligonucleotide using:

• Phosphorimaging – High-resolution detection
• Autoradiography – Traditional film-based detection

Why Polynucleotide Kinase and γ-32P ATP Are Used

 Polynucleotide kinase has high specificity for 5′-end hydroxyl groups.
 γ-32P ATP provides high sensitivity due to the radioactive label.
The labeled oligonucleotide is stable and retains high signal strength.

Explanation of Other Options

B. R and S

• DNA-dependent RNA polymerase – This enzyme synthesizes RNA from a DNA template, but it is not involved in direct oligonucleotide labeling.
• DNA ligase – This enzyme joins two DNA strands by forming a phosphodiester bond, but it cannot attach a radiolabel to the oligonucleotide.

C. P and R

• γ-32P ATP can provide a radioactive label, but DNA-dependent RNA polymerase cannot transfer this label to the oligonucleotide.

D. Q and S

• Polynucleotide kinase can phosphorylate oligonucleotides, but DNA ligase does not participate in direct labeling.
• DNA ligase is used for joining nucleic acid fragments, not for labeling.

Applications of Labeled Oligonucleotides

Southern and Northern Blotting

• Labeled oligonucleotides can serve as probes for detecting specific DNA or RNA sequences.

DNA Footprinting

• Helps identify protein-binding sites on DNA.

Electrophoretic Mobility Shift Assay (EMSA)

• Labeled oligonucleotides are used to detect DNA-protein interactions.

Real-Time PCR

• Labeled probes enable quantitative analysis of gene expression.

Advantages of Polynucleotide Kinase and γ-32P ATP Labeling

High specificity – Target-specific binding of labeled oligonucleotides.
 High sensitivity – Radiolabeled ATP allows detection of very low target concentrations.
 Versatility – Suitable for both DNA and RNA.

Limitations

Requires handling of radioactive material under strict safety protocols.
 γ-32P ATP has a short half-life (~14 days), limiting long-term use.
Radiolabeled oligonucleotides require proper disposal procedures.

Alternative Labeling Methods

Safety Considerations for Radioactive Labeling

 Work with γ-32P ATP in a designated radioactive workspace.
 Wear protective gloves, lab coat, and eye protection.
 Dispose of radioactive waste in compliance with local regulations.
Monitor radiation exposure using a Geiger counter.

Conclusion

Labeling oligonucleotides using polynucleotide kinase and γ-32P ATP is a highly effective method for molecular biology applications, offering high sensitivity and specificity. Polynucleotide kinase transfers the radioactive phosphate from γ-32P ATP to the 5′ end of the oligonucleotide, allowing easy detection in various assays. This technique remains a gold standard for high-sensitivity DNA and RNA analysis.