Which one of the following can be extended by Klenow enzyme upon addition of dNTPs and Mg 2+?
1. restriction fragment with a 5′ overhang
2. restriction fragment with a 3′ overhang
3. single-stranded DNA
4. restriction fragment with blunt ends

Introduction to Klenow Enzyme

Klenow enzyme is a powerful tool used in molecular biology for DNA manipulation. It is a fragment of the DNA polymerase I enzyme, which is originally derived from E. coli. Klenow polymerase is widely used for various tasks such as filling in gaps, extending DNA fragments, and synthesizing complementary strands.

Klenow Enzyme and Its Ability to Extend DNA

The Klenow enzyme has two major activities:

• Polymerase activity: It can add nucleotides to a 3′ end of a DNA strand, extending the strand.

• Exonuclease activity: It has the ability to remove nucleotides from the 3′ end, which is useful for correcting mistakes in the DNA sequence.

However, Klenow enzyme cannot add nucleotides to a 5′ overhang without the proper conditions. When the enzyme is provided with dNTPs (deoxyribonucleotide triphosphates) and Mg²⁺ ions, it can extend a restriction fragment with a 5′ overhang.

Why Klenow Enzyme Extends a 5′ Overhang

1. 5′ Overhangs: Klenow enzyme works by filling in the gap on the 3′ end of a DNA fragment with a 5′ overhang. This type of overhang provides the required template for Klenow polymerase to add complementary nucleotides to the exposed 3′ end, allowing for strand extension.

2. 3′ Overhangs: The enzyme does not extend DNA fragments with a 3′ overhang as efficiently because the 3′ overhang cannot be extended by the polymerase activity of Klenow without additional processing.

3. Blunt Ends and Single-Stranded DNA: Klenow enzyme is less effective at extending blunt-ended DNA or single-stranded DNA. These structures do not have the necessary overhangs for the enzyme to bind and initiate extension.

Applications of Klenow Enzyme in Molecular Biology

Klenow enzyme is a valuable tool in various genetic research and biotechnology applications:

• DNA Cloning: Klenow enzyme is used to fill in overhangs after restriction digestion, creating blunt ends for ligation into vectors.

• DNA Repair: It can be used to repair and fill in DNA gaps during PCR or other DNA amplification processes.

• Site-directed Mutagenesis: Klenow enzyme can be used to insert specific mutations into DNA sequences during cloning experiments.

Conclusion

Klenow enzyme is an essential tool for extending restriction fragments with 5′ overhangs. By adding dNTPs and Mg²⁺, it efficiently extends these fragments, which is crucial for various molecular biology applications, including cloning and DNA repair. Understanding the enzyme’s capabilities is key to utilizing it effectively in experiments that require the manipulation of DNA sequences.