Klenow fragment: Definition and Applications
Definition: It is the large fragment of DNA polymerase I enzyme of E.coli produced by the proteolytic cleavage of DNA pol I by protease subtilisin.
Refer this figure for clear understanding. On the right side is the DNA Polymerase I before treating with protease. On the left side, upon treatment with protease, two fragments are formed, a large fragment called the klenow fragments and a small fragment with 5'→ 3' exonuclease activity.
This klenow fragment lacks 5'→ 3' exonuclease activity.
It has 5'→ 3' polymerase activity
It has 3'→ 5' exonuclease activity for proof reading activity
The other small fragment has 5'→ 3' exonuclease activity, but lacks 5'→ 3' polymerase activity and 3'→ 5' exonuclease activity of klenow fragment.
- Synthesis of second strand on cDNA
- DNA sequencing by the Sanger dideoxy method
- Fill-in of 5´ overhangs to form blunt ends
- Removal of 3´ overhangs to form blunt ends
- Preparation of radioactive DNA probes.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. (2nd ed.), 5.40-5.43. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.