KLENOW FRAGMENT (DNA POL I -LARGE FRAGMENT )

The Klenow fragment is a large protein fragment  produced when DNA polymerase I from E. coli is enzymatically cleaved by the protease subtilisin. 

The  cleavage results in  1. Small fragment of  322 amno acids having 5'-3' exonuclease activity .
                                   2.Large fragment of 604 amino acids having DNA polymerization and 3'-5' exonuclease activity .

The larger fragment retains the 5' → 3' polymerase activity and the 3’ → 5’ exonuclease activity for removal of precoding nucleotides and proofreading, but loses its 5' → 3' exonuclease activity.

The other smaller fragment formed when DNA polymerase I from E. coli is cleaved by subtilisin retains the 5' → 3' exonuclease activity but does not have the other two activities exhibited by the Klenow fragment (i.e. 5' → 3' polymerase activity, and 3' → 5' exonuclease activity).

The elimination of exonuclease activity makes Exo- Klenow Fragment the enzyme of choice for isotopic or biotin labeling of DNA probes by the random primed method and for DNA sequencing by the sanger dideoxy method.

Applications -

DNA blunting - By filling-in 5'-overhangs with unlabeled or labeled dNTPs

cDNA strand synthesis 

Generate single-stranded DNA probes using random primers

Site-directed DNA mutagenesis using synthetic oligonucleotides

Dideoxy DNA sequencing of single- or double-stranded DNA templates

3’→5’ exonuclease activity can blunt a 3’-overhang.

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