What are the differences between DNA polymerase 1 and Klenow fragment?
The key difference between Klenow fragment and DNA polymerase 1 is that Klenow fragment is a large portion of DNA polymerase 1 which lacks 5′ to 3′ exonuclease activity while DNA polymerase is an enzyme of E. coli which has all three domains including 5′ to 3′ exonuclease activity.
Does Klenow have exonuclease activity?
The Klenow fragment retains the polymerizing activity and the 3′ → 5′ exonuclease of the holo-enzyme but lacks its powerful 5′ → 3′ exonuclease activity. These enzymes and their applications in molecular cloning are introduced here.
Why is Klenow fragment used in Sanger sequencing?
The Klenow fragment is extremely useful for research-based tasks such as: Synthesis of double-stranded DNA from single-stranded templates. Filling in receded 3′ ends of DNA fragments to make 5′ overhang blunt. Digesting away protruding 3′ overhangs.
Is DNA polymerase I (Klenow) active in nebuffers?
DNA Polymerase I, Large (Klenow) Fragment is also active in all four NEBuffers and T4 DNA Ligase Reaction Buffer when supplemented with dNTPs. Jacobsen, H., Klenow, H. and Overgaard-Hansen, K. (1974).
What does Klenow stand for?
DNA Polymerase I, Large (Klenow) Fragment is a proteolytic product of E. coli DNA Polymerase I which retains polymerization and 3’→ 5′ exonuclease activity, but has lost 5’→ 3′ exonuclease activity (1). Klenow retains the polymerization fidelity of the holoenzyme without degrading 5′ termini.
What is Klenow polymerization fidelity?
Klenow retains the polymerization fidelity of the holoenzyme without degrading 5′ termini. An E. coli strain that contains the E. coli polA gene that has had its 5’→3′ exonuclease domain removed.
What is the reaction volume of Klenow fragment for DNA sequencing?
When Klenow Fragment (3´→ 5´ exo-) is used to sequence DNA using the dideoxy method of Sanger et al., 1 unit/5 µl reaction volume is recommended. Derbyshire, V. et al. (1988). Science. 240, 199-201. Sanger, F. et al. (1977).