Klenow fragment

PolymeraseDomains

Klenow Fragment is a large fragment of the DNA polymerase I enzyme that is used extensively in molecular biology for adding nucleotides to the 3' end of a DNA strand. The fragment is named after Danish biochemist Hans Klenow who first isolated it in the early 1970s. Unlike the complete DNA polymerase I enzyme, which possesses both 5'→3' polymerase activity and 5'→3' and 3'→5' exonuclease activities, the Klenow Fragment has only the 5'→3' polymerase activity and the 3'→5' exonuclease activity, making it useful for certain DNA sequencing and DNA labeling techniques.

Structure and Function

The Klenow Fragment is derived from the full-length DNA polymerase I by proteolytic cleavage, which removes the 5'→3' exonuclease domain of the enzyme. This leaves the 5'→3' polymerase and 3'→5' exonuclease activities intact. The 3'→5' exonuclease activity provides a "proofreading" function, allowing the enzyme to remove incorrectly incorporated nucleotides, thereby increasing the fidelity of DNA synthesis.

Applications in Molecular Biology

The Klenow Fragment has several applications in molecular biology, including:

• DNA sequencing: It is used in some methods of DNA sequencing, where its ability to synthesize DNA in the 5'→3' direction can be harnessed to extend primers annealed to a template strand.
• DNA labeling: The enzyme can be used to add labeled nucleotides to the 3' end of DNA fragments, which is useful in various types of DNA probes for hybridization experiments.
• Blunting of DNA ends: The Klenow Fragment can be used to convert sticky ends of DNA fragments into blunt ends, facilitating the cloning of DNA fragments into vectors that only accept blunt-ended fragments.

Advantages and Limitations

The Klenow Fragment offers high fidelity in DNA synthesis due to its 3'→5' exonuclease activity. However, its use is limited by its sensitivity to heat denaturation and the inability to displace strands during synthesis, unlike some other DNA polymerases such as Taq polymerase.

See Also

• DNA polymerase
• Molecular cloning
• Polymerase chain reaction (PCR)
• Taq polymerase