DNA Polymerase I Large (Klenow) Fragment Protocol
Literature # 9PIM220
DNA Polymerase I Large (Klenow) Fragment consists of a single polypeptide chain (68kDa) that lacks the 5´->3´ exonuclease activity of intact E. coli DNA polymerase I, but retains its 5´->3´ polymerase, 3´->5´ exonuclease and strand displacement activities. The 5´->3´ polymerase activity of Klenow Fragment can be used in the following applications: a) to fill in 5´-protruding ends with unlabeled or labeled dNTPs; b) to sequence single or double-stranded DNA templates; c) for in vitro mutagenesis experiments using synthetic oligonucleotides; d) for cDNA second strand synthesis; and e) to generate single-stranded DNA probes.
The 3´->5´ exonuclease activity can be used to generate blunt ends from a 3´-overhang. Klenow Fragment, Exonuclease Minus, which is deficient in both the 5´->3´ and the 3´->5´ exonuclease activities, is primarily used for sequencing of DNA templates.