Footprinting là gì


Footprinting

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?

The procedure:

  • 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.
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