Which of the following methods are used to label the double stranded DNA?
(A) 5' - end labelling with polynucleotide kinase
(B) 3' - end labelling with polynucleotide kinase
(C) 5' - end labelling with terminal transferase
(D) 3' - end labelling with terminal transferase
Choose the correct answer from the options given below:
Show Hint
Remember the enzyme targets:
- Kinase $\rightarrow$ adds phosphate to 5'-OH.
- Terminal Transferase $\rightarrow$ adds nucleotides to 3'-OH.
Step 1: Understanding the Concept:
End-labeling is a molecular biology technique used to introduce a radioactive (e.g., $^{32}\text{P}$) or fluorescent tag at either the 5' or 3' end of a DNA fragment.
This is accomplished using highly specific enzymes that target these termini. Detailed Explanation:
Let us analyze the enzymatic mechanisms of the options:
- Polynucleotide Kinase (T4 PNK): This enzyme catalyzes the transfer of the $\gamma$-phosphate group from ATP (often $^{32}\text{P}$-labeled) to the 5'-hydroxyl terminus of double-stranded or single-stranded DNA or RNA.
It does not label the 3' end. Thus, (A) is correct and (B) is incorrect.
- Terminal Deoxynucleotidyl Transferase (TdT): This is a template-independent DNA polymerase that catalyzes the repetitive addition of deoxynucleotides (often labeled) to the 3'-hydroxyl terminus of a DNA molecule.
It cannot add nucleotides to the 5' end. Thus, (D) is correct and (C) is incorrect.
Therefore, the correct methods are 5'-end labeling with polynucleotide kinase (A) and 3'-end labeling with terminal transferase (D). Step 2: Final Answer:
The correct option is (A), representing (A) and (D) only.
