Question

Briefly describe Nick Translation technique.

Hint:

Because DNA Polymerase I proofreads while polymerising, Nick Translation produces highly accurate, uniform-length labeled probes without denaturing the double-stranded template.

Answer 1

Step 1: Definition and Purpose
Nick translation is an in vitro enzymatic process used to label DNA molecules with radioactive or fluorescent nucleotides. This generates labeled DNA probes for hybridisation techniques like FISH, Southern blotting, or Northern blotting.

Step 2: Procedure
1. Nicking: The double-stranded DNA template is treated with a very low concentration of the endonuclease DNase I. This introduces random single-stranded breaks (nicks) in the phosphodiester backbone, exposing a free 3'-OH group and a 5'-phosphate group.
2. Enzymatic Action: E. coli DNA Polymerase I is added to the reaction along with dNTPs containing labeled nucleotides (such as biotinylated, digoxigenin-conjugated, or fluorescent dUTPs).
3. Exonuclease Activity: DNA Polymerase I uses its 5' to 3' exonuclease activity to degrade the existing, unlabeled nucleotides starting from the nick in the 5' to 3' direction.
4. Polymerase Activity: Simultaneously, the polymerase uses its 5' to 3' polymerase activity to add new, labeled nucleotides to the 3'-OH end, using the complementary strand as a template.
5. Nick Translation: The position of the nick is "translated" along the DNA strand, producing a highly labeled DNA probe.
Final Answer: Nick translation uses DNase I to introduce single-stranded nicks in DNA. DNA Polymerase I then uses its 5' to 3' exonuclease activity to remove unlabeled nucleotides from the nick, while simultaneously polymerising new, labeled nucleotides in their place.