Labeling 3′ Termini of Double-Stranded DNA Using the Klenow Fragment of E. coli DNA Polymerase I

The Klenow fragment, which retains the template-dependent deoxynucleotide polymerizing activity and the 3′ → 5′ exonuclease of the holo-enzyme but lacks its powerful 5′ → 3′ exonuclease activity, is used to fill recessed 3′ termini of dsDNA. In this protocol, fragments suitable as templates for the end-filling reaction are produced by digestion of DNA with an appropriate restriction enzyme. The Klenow enzyme is then used to catalyze the attachment of dNTPs to the recessed 3′-hydroxyl groups.