Klenow fragment Large fragment produced by Subtilisin mediated proteolytic cleavage of E.Coli DNA polymerase I. Proteolysis removes the 5' -->3' exonuclease activity from N-terminal. Klenow fragment - Functions : Remove 3' overhang Fills 5' overhangs Synthesis of double-stranded DNA from single-stranded templates Preparation of radioactive DNA probes Was used in PCR