532
chapter 23
Structure and Properties of DNA
(a) Cuts on axis
of symmetry
.........
_
!
_____________
__________t c !g a .
...................
_______ A G ,C T ________
Î
Separation of
fragments
------------ 3' î
5'.
.................
_______ T C
.
G A ________
...............
A G
,
C T .
...............
------------ 5'
3'------------
Flush-end molecules
(b) Cuts symmetrically placed
around axis of symmetry
______ 1_____________
__________g a a It t c
....................
_______CTT, A A G _______
t~ ~
Separation of
fragments
-----------3’
5'-------------------
_______ G
A A T T C .
...............
_______CTTAA
G _______
Cohesive-end molecules
FIGURE 23-11
Two types of cuts made by restriction enzymes. The arrows indicate the
cleavage sites. The dashed line is the axis of symmetry of the sequence.
Figure 23-12a shows the positions of restriction sites
in bacteriophage lambda (À) DNA for the restriction
enzymes EcoRI and BamHI. This arrangement of restric-
tion sites is known as a
restriction map.
The family of
fragments generated by one or more restriction enzymes
is detected by agarose gel electrophoresis that separates
DNA molecules according to their molecular weights
(Figure 23-12b).
23.7
Diagnostic and Clinical
Applications of DNA
DNA Probes
The ability to isolate specific fragments of DNA contain-
ing known sequences of genes gives rise to
DNA probes
that can be used for a variety of diagnostic, forensic, and
therapeutic purposes. DNA probes can be labeled with ei-
ther radioactive or nonradioactive markers. A DNA probe
has a strong interaction with (ideally) a specific DNA tar-
get and can be detected after the interaction. DNA probes
consisting of
2 0
bases or fewer usually will have a unique
target even in a large set of DNA molecules. The proba-
bility that any base will follow any other base in DNA is
one in four or 0.25. Therefore, the probability of a specific
sequence of 20 bases occurring in a DNA molecule by
chance is 0.2520, a vanishingly small number.
The use of DNA probes in various aspects of medical
diagnostics is increasing rapidly. DNA probes can be used
to identify infectious agents if sequences specific to dif-
ferent pathogens are known. Identification of a pathogen
by DNA probes can be done in hours as compared to days
or weeks by conventional culturing of microorganisms.
DNA probes are now used routinely to detect the pres-
ence of mutant alleles in fetal cells obtained by am-
niocentesis, as well as in cells removed from affected
adults or carriers. Many inherited disorders, such as
sickle cell disease, cystic fibrosis, Huntington’s disease,
Duchenne’s muscular dystrophy, and dozens of other
Mendelian (single-gene) disorders, can now be diagnosed
in fetuses and adults. In addition to inherited disorders,
DNA probes are used to detect the presence of active onco-
genes or inactive tumor suppressor genes in cancerous tis-
sues removed from patients (Chapter 26).
Southern Blot Analysis
The
Southern blot
(named for its inventor, E. M. Southern)
is a method for hybridizing one or more labeled DNA
probes to a large number of DNA fragments and discrimi-
nating among them. The procedure depends on the ability
of denatured DNA single strands to bind tightly to nitro-
cellulose under certain conditions.
The Southern blot procedure is outlined schematically
in Figure 23-13. The DNA to be investigated is digested
with several restriction enzymes to generate DNA frag-
ments of varying sizes which are then separated by agarose
gel electrophoresis. After the fragments are separated, the
gel is immersed in a denaturing solution that converts all
DNA to single strands. Then the gel, which is in the form
of a broad, flat slab, is placed on top of filter paper sup-
ported by a glass plate. A nitrocellulose filter is placed
over the gel and covered with another sheet of filter paper.
Finally, paper towels and weights are placed on top of
the gel.
The glass plate supporting the filter papers and gel is ad-
justed so that the ends of the filter paper are suspended in
buffer and act as wicks. The buffer moves upward through
the gel and into the wad of paper towels that act to absorb
the buffer. The DNA migrates from the gel to the nitrocel-
lulose filter that binds the single-stranded DNA fragments.
