Footprinting was first used by Galas^67 to determine the binding sequence for the lacrepressor protein
that established the operator sequence: d(CACCTTAACACTAACCTCTTGTTAAAG)-5. It is now possi-
ble to identify stronger and weaker protein binding and to differentiate between affinities for each of the
two DNA strands.
Since protein binding in vitromay not accurately reflect binding-site occupancy in the cell nucleus,
methods have been developed for DNA footprinting in vivo. The GA-LMPCR in vivofootprinting system
employs dimethyl sulfate(0.3–1.5%) to methylate nuclear DNA in whole cells suspended in phosphate
buffer. Methylation occurs mainly at guanine N-7 in the major groove (Section 8.5.3) with further methyl-
ation at adenine N-3 in the minor groove. Incubation of the protein-free genomic DNA at 90°C and pH 7.0
for 15 min followed by treatment with 1 M NaOH for 30 min at 90°C leads to specific cleavage at methyl-
ated G and A sites (Maxam–Gilbert G A procedure) (Section 5.1.1) Guanine-specific cleavage can also
be accomplished by piperidine treatment of methylated DNA. The cleaved strands are then amplified
using ligation-mediated PCR and analysed by PAGE, as above. Such methods have been used for the
detection of upstream regulatory sequences, known as locus control regions.68,69
For some purposes, cleavage of the DNA is better achieved by chemical means and one of the most suc-
cessful reagents has been the hydroxyl radical: Fenton’s reagent. The cleavage system used is ferrous
ammonium sulfate (1 mM) in conjunction with ascorbic acid (10 mM) and hydrogen peroxide (0.3%) at
room temperature for 2 min. This works by generating hydroxyl radicals that abstract a hydrogen atom
from the deoxyribose leading to phosphate diester cleavage at that residue (Section 8.9.1).^70
In addition to its use in studies of protein binding to DNA, footprinting has been widely employed, for
example, for investigating the selectivity of drug binding to DNA (Chapter 9) and for conformational
analysis of triple helix formation.^71
204 Chapter 5
Figure 5.30 Scheme illustrating DNA footprinting for lac repressor protein binding to dsDNA containing the lac
operator sequence. DNase I cuts DNA molecules randomly. Only one strand is 5-end labelled with^32 P.
For polyacrylamide gel electrophoresis see Section 11.4.3