Klenow fragment

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The larger of the two protein fragments of DNA polymerase I from Escherichia coli , which are formed after enzymatic cleavage with subtilisin , is referred to as the Klenow fragment , also known as the Klenow enzyme . It still has the 5 '→ 3' polymerase activity and the 3 '→ 5' exonuclease activity ( proof reading ), but no longer the 5 '→ 3' exonuclease activity of DNA polymerase I. The protein fragment was first published in 1970 described by the Danish biochemist Hans Klenow.

use

The Klenow fragment is used in molecular biology for various purposes:

  • Synthesis of double-stranded DNA (polymerase function)
  • the Klenow fragment was the original enzyme used to amplify DNA using PCR before being replaced by thermostable DNA polymerases such as Taq polymerase . The Klenow enzyme is not thermally stable and had to be added again in each cycle.
  • Filling in 5 'protruding, single-stranded DNA sequences according to restrictions (polymerase function)
  • Degradation of 3 'protruding, single-stranded DNA sequences according to restrictions (exonuclease function)

After the DNA has been cut with restriction enzymes, these activities are used to produce so-called blunt ends , which can be linked by means of T4 ligase (e.g. during cloning ). In addition to the DNA template, the 4 nucleotide triphosphates dGTP, dATP, dTTP and dCTP are offered in the magnesium-dependent reaction. The polymerase activity is also used to mark ( end labeling ) DNA fragments with radioactive phosphorus isotope 32 P by replacing a dNTP, for example dATP by α- 32 P-ATP and as an enzyme in random priming . Further applications are DNA sequencing with the Sanger dideoxy method and second strand synthesis in the cloning of cDNAs . 

Individual evidence

  1. ↑ Klenow, H. & Henningsen, I. (1970): Selective elimination of the exonuclease activity of the deoxyribonucleic acid polymerase from Escherichia coli B by limited proteolysis. In: Proc. Natl. Acad. Sci. USA 65 (1): 168-175, PMID 4905667 , PMC 286206 (free full text).
  2. ↑ Saiki, RK et al. (1985): Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. In: Science. 230 (4732): 1350-1354, PMID 2999980 .
  3. ↑ Saiki, RK et al. (1988): Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. In: Science. 239 (4839): 487-491, PMID 2448875 .