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.

References

NEB Technical Resources
Technical Resources, PDB
Other Internet Sources