DNA Footprinting - Definition, Principle and Procedure

Definition: An in-vitro technique to find out protein binding regions on a DNA molecule. The technique is also called as DNAse I footprinting.

Thousands of proteins (enzymes) are interacting with DNA in the nucleus for regulating activities like replication, transcription, translation etc. The technique to find out this protein binding regions in DNA molecule is called DNA footprinting.

Examples:

To find out regions in a gene where transcription factors (proteins) binds (that is control elements like operators, promoters etc) and initiates transcriptions.

To find out Hormone response elements (HREs) that is specific sequences where hormone receptor complexes bind.

Principle: In this technique, nucleases like DNAse I is used which will degrade DNA molecule. Nucleases cannot degrade DNA if it is bounded by a protein. Thus that region is protected from degradation by nucleases. This protected DNA region is called the foot print.

Procedure:

1.Radioactive 5’ end labeling of the DNA suspected to contain one or more protein binding sites

2.Two DNA samples; one incubated with suspected protein and other without the protein
3.The DNA is treated with a nuclease such as DNAse I, that digests only unprotected DNA

DNAse I is used under specific digestion condition to obtain one cut or hit per molecule, resulting in a complete base ladder (one base difference) when electrophoresed in 6-8% polyacrylamide gel.

4.The resulting products are separated on a Polyacrylamide gel electrophoresis (PAGE)
In DNA sample with protein, protein binding regions are protected from degradation by DNAse I
5.X-ray film exposure and autoradiography.

Comparison of both samples reveals foot prints or protein binding sites.

In the figure

• DNA Sample A without protein: consistent degradation by DNAse I resulting in a continuous ladder
• DNA Sample B with protein (lac repressor): Interrupted degradation by DNAse I as protein or lac repressor bound regions are protected from cleavage by DNAse I. This protected DNA region is called the “DNA foot print”

Application: To study DNA-protein interaction