Klenow-Fragment

As Klenow fragment, and Klenow enzyme, the larger of the two protein fragments of the DNA polymerase I from Escherichia coli is known to occur by enzymatic digestion with subtilisin. It still possesses the 5 '→ 3' polymerase activity and 3 '→ 5' exonuclease activity ( proofreading ), but not the 5 '→ 3' exonuclease activity of DNA polymerase I. The protein fragment was in 1970 described by the Danish biochemist Hans Klenow.

Use

Klenow fragment is used in molecular biology for a variety of purposes:

• Synthesis of double-stranded DNA (polymerase function)

The Klenow fragment was the original enzyme was used for the amplification of DNA by means of PCR, before being replaced by thermostable DNA polymerases such as Taq polymerase.

• Filling of the 5'- protruding, single-stranded DNA sequences by restrictions ( polymerase function)
• Degradation of the 3'- protruding, single-stranded DNA sequences by restrictions ( exonuclease )

These activities serve after cutting DNA with restriction enzymes for the production of so-called blunt ends that can be joined using T4 ligase (eg cloning ).