Practice question

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.