Footprint analysis

Introduction

• Advantage: can see the binding site on DNA
• Disadvantage
  • relatively difficult to perform than filter binding assay or EMSA
  • protect a large region which may include many contact points

Rationale

• A DNA binding protein will protect the binding site from DNaseI attack

Methods

• Mix protein and DNA to form complex
• Add DNaseI to cleave DNA
• Kill the enzyme and purify DNA
• Analyze cleavage product on gel

This experiment is not trivial

• DNaseI only cleave one strand on a double stranded DNA
• To what extent the DNA should be cleaved? The cutting should be rare. Most of the DNA should be either be cut once or not cut.

  If the end-labeled DNAs were cut twice, the sequencing gel will be difficult to interprete. Besides, it will be difficult to judge whether we are observing the protection of the normal complex or the protein protection of a short DNA fragment. A simple to judge the extent of cutting is that there are about 90% of uncut DNA left on the top part of a gel.

• There are two ways to cut the DNA:
  • More enzyme and short digestion time
  • Less enzyme and long digestion time

  The DNA-protein complex may dissociate and reassociate constantly. If the DNaseI cut the protein region, these two fragment will no longer be able to interact with protein. As a result, the protection effect will not be observed clearly. To overcome this problem, the cutting time should be smaller than the halflife of a typical DNA-protein complex, which is about 10 min.

• Why the protein fails to bind cleaved DNA?