Gene Cloning

The Analysis of the Regulation of Gene Expression 341
Test
Control
Enhancer/
promoter
element
Isolate chromatin
template and treat
with DNAse I
Isolate chromatin
template
Probe
DNAse I
Purify DNA and cut with
restriction enzyme
Probe
RE RE RE RE
Southern
analysis
M A B
Distance between restriction
enzyme sites
Distance between one
restriction enzyme site
and site of DNAse I cleavage
Figure 11.14 DNAse I hypersensitivity assay. Intact chromatin is isolated
from cells expressing the gene of interest and treated with DNAse I, which
cleaves the DNA where there are no nucleosomes or where the nucleosome
structure is altered. The DNA is purified and analyzed by Southern analysis using
DNA that has not been treated with DNAse I as a control (lane B). DNAse I
cleavage results in detection of a shorter fragment in the Southern blot (lane A),
the difference in size can be used to map the position of the DNAse I
hypersensitive site relative to the position of the restriction enzyme recognition
sites. DNA standards are used to calibrate the gel (M).
more transcription factors (Figure 11.15a). As you will be aware, the migration
of a linear DNA fragment during gel electrophoresis is mainly dependent
on the length of the DNA fragment. It is possible to analyze DNA–
protein complexes using gel electrophoresis and, since these complexes
have a higher mass than the free DNA, they will migrate at a slower rate