- A 68kDa C-terminal fragment of E. coli DNA Polymerase I
- The Klenow fragment is a large proteolytic fragment of DNA polymerase I from Escherichia coli that retains its polymerase and 3′→5′ exonuclease (proofreading) activities, but lacks the 5′→3′ exonuclease activity of the full-length enzyme.
- It was originally produced by proteolytic digestion with subtilisin, which cleaves the N-terminal portion responsible for 5′→3′ exonuclease function, yielding a fragment approximately 68 kDa in size.
- Due to its retained ability to synthesize DNA in the 5′→3′ direction and to correct errors via 3′→5′ exonuclease proofreading, the Klenow fragment is commonly used in molecular biology applications requiring precise DNA polymerase activity without degradation of DNA from the 5′ end. It is frequently employed in blunt-ending of DNA fragments, fill-in reactions of 5′ overhangs, random-primed DNA labeling, and second-strand cDNA synthesis.
- In its wild-type form, the Klenow fragment is able to remove mismatched nucleotides at the 3′ ends, thereby improving fidelity during synthesis. A mutant version, known as the exo– Klenow fragment, has the 3′→5′ exonuclease activity inactivated, making it suitable for applications where proofreading is not desired, such as when incorporating modified nucleotides or labeling.
- Although largely replaced by more efficient enzymes like Taq polymerase or Q5 polymerase in routine PCR, the Klenow fragment remains valuable in specialized procedures where controlled DNA extension without strand degradation is essential.
- In summary, the Klenow fragment of E. coli DNA polymerase I is a versatile enzyme tool in DNA manipulation, providing accurate polymerase activity without 5′→3′ exonuclease function, and is particularly useful in cloning and labeling workflows.
Applications
- Labeling of oligonucleotide
