Which end of DNA is typically labeled during restriction mapping?
Restriction mapping tracks fragments originating from a defined terminus to deduce site order, so terminal labeling is essential. Restriction enzymes producing 5' overhangs expose a recessed 3'-OH and a free 5'-phosphate, ideal substrate for T4 polynucleotide kinase to add 32P or for Klenow fragment to fill in with radioactive dNTPs. Internal loci remain phosphodiester-linked without free ends, blunt termini incorporate label poorly, and 3'-only labeling lacks efficiency. Therefore labeling free 5' cohesive ends creates a single radioactive reference point for stepwise fragment analysis.
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.