Which of the following is a method of investigating the sequence specificity of DNA- binding proteins in
vitro?
1. gene targeting
2. DNA footprinting
3. polymerase chain reaction
4. Southern hybridization

Which of the following is a method of investigating the sequence specificity of DNA-binding proteins in vitro?

1. Gene targeting

2.  DNA footprinting

3. Polymerase chain reaction

4. Southern hybridization

Why DNA Footprinting Is the Correct Answer

DNA footprinting is a biochemical technique used in vitro to identify the specific DNA sequences bound by proteins. It reveals which regions of a DNA molecule are protected by a bound protein from enzymatic cleavage or chemical modification.

How it works:

1. A DNA fragment is labeled (usually at one end).

2. It’s incubated with and without a DNA-binding protein.

3. The DNA is then treated with a DNA-cleaving agent (e.g., DNase I).

4. In the presence of the protein, the bound region is protected from cleavage.

5. When run on a gel, this “missing band” shows the footprint—the area where the protein binds.

This allows researchers to determine the exact sequence that the protein recognizes and binds to.

Why the Other Options Are Incorrect:

• 1. Gene targeting
   Involves altering or knocking out genes in living organisms. It’s in vivo, not used to analyze protein-DNA interactions in vitro.

• 3. Polymerase chain reaction (PCR)
   PCR is used to amplify DNA sequences, not to study protein binding specificity.

• 4. Southern hybridization
   Used to detect specific DNA sequences on a gel. It doesn’t study protein-DNA binding or sequence specificity.

Conclusion: DNA Footprinting Reveals the Hidden Language of DNA-Protein Binding

DNA footprinting is the go-to method for identifying the exact DNA sequences recognized by binding proteins in vitro. It’s a cornerstone in molecular biology for studying transcription factors, repressors, and other regulatory proteins.

Perfect for researchers and students aiming to understand how genes are regulated at the molecular level.