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Wednesday, November 4, 2015

Klenow Fragment - Sturctural Analysis, Comaprison and Applications

What is Klenow Fragment??

  • Klenow Fragment is the proteolytic cleavage product of DNA Polymerase I. 
  • Klenow Fragment is generated when DNA polymerase I is cleaved using protease subtilisin. 
  • Klenow fragment retains 5’-3’ polymerase activity and 3’-5’ proof reading activity. 

Structure of DNA Polymerase 

DNA Polymerase has three domains:

1. Polymerase domain.
2. Proofreading domain.
3. Exonuclease domain.

Klenow Fragment has only 2 domains:

1. Polymerase domain 
2. Proofreading domain

Klenow Fragment lacks 5'-3' exonuclease domain.

Structural Comparison of DNA polymerase and Klenow Fragment

Applications / Advantages of Klenow Fragment
  • DNA sequencing by the Sanger dideoxy method.
  • Fill-in of 5´ overhangs to form blunt ends.
  • Removal of 3´ overhangs to form blunt ends.
  • Second strand cDNA synthesis.
  • Second strand synthesis in mutagenesis protocols.

NEB Technical Resources
Technical Resources, PDB
Other Internet Sources