Footprinting is a method for determining the exact DNA sequence to which a particular DNA-binding protein binds. Examples:
How, for example, does one determine the DNA sequence to which the lac repressor binds?
- Clone a piece of DNA that contains the operator site to which the repressor binds.
- Label one end of the DNA molecules with a radioactive molecule, e.g. radioactive ATP.
- Digest the DNA with DNase I.
- DNase I cuts DNA molecules randomly (in contrast to restriction enzymes that cut where they find a particular sequence)
- Choose such gentle conditions that most molecules will be cut only once.
- The result will be a mixture of radioactive fragments of varying length, with the smallest increment in length represented by a single nucleotide.
- Separate the fragments by electrophoresis.
- Binding of the lac repressor to the sequence of 24 base pairs in the operator prevents DNase I from attacking that region of the molecule.
- When the fragments are separated by electrophoresis, those representing the lengths covered by the repressor will be missing from the autoradiogram.
- The resulting gap is the "footprint".
- The same sample of DNA (unprotected by the repressor) is subjected to normal DNA sequencing and the resulting ladder aligned with the footprint autoradiogram.
- The exact sequence of bases in the lac operator can then be read directly because they represent the rungs of the ladder missing in the footprint.
|Link to a discussion of chromatin immunoprecipitation (ChIP) — another method by which the DNA sequence to which a particular DNA-binding protein binds can be determined.
18 April 2014