Klenow Fragment (exo–), corresponds to the large (Klenow) fragment of E. coli DNA polymerase I, which due to truncation lacks 5’ → 3’ exonuclease activity, and that was mutated to inactivate the inherent 3′ → 5’ proofreading activity, while retaining polymerase activity. The enzyme displays moderate strand displacement activity and is of particular interest to generate probes using random primers, dideoxy sequencing and random priming labelling. The enzyme is also effective to fill-in 5′-overhangs of dsDNA. Note that Klenow Fragment (exo–) is not recommended for DNA blunting reactions prior to DNA ligation since it frequently adds one or more extra nucleotides to the 3′-terminus of blunt-end DNA substrates in a non-template directed fashion.
Applications:
– Fill-in 5′-overhangs of dsDNA
– Generates probes using random primers
– Dideoxy sequencing
– Random priming labelling