section 23.7
Diagnostic and Clinical Applications of DNA
533
EcoRI
EcoRI
B am H I
(b)
F I G U R E 2 3 - 1 2
(a) Restriction maps of
X
DNA for EcoRI and BamHI nucleases. The vertical bars indicate the sites of cutting. The top
numbers indicate the percentage of the total length of DNA measured from the gene A end of the molecule. The bottom
numbers are the lengths of each fragment, again expressed as percentage of total length, (b) Gel electrophoretogram of
EcoRI and BamHI restriction enzyme digests of
X
DNA. The bands labeled “cohered ends” contain molecules
consisting of two terminal fragments, joined by the normal cohesive ends of
X
DNA. Numbers indicate fragments in
order from largest 1 to smallest
6
. Bands 5 and
6
of the BamHI digest are not resolved.
- DNA restriction fragments
Agarose get electrophoresis
I
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n il
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f
Fragments separated by size
•Weight
Paper towels
Nitrocellulose filter
Gel
Transfer of
DNA fragments
from gels to
nitrocellulose
filter
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unn
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,
^Nitrocellulose filter
"with DNA fragments
Radioactive DNA probe hybridized
to fragments on filter
_ Autoradiograph showing
positions of hybrid DNA
F I G U R E 2 3 - 1 3
Procedure for a Southern blot. This analysis allows detection of a specific
fragment of DNA by hybridization of a radioactive probe with a
complementary sequence.
The nitrocellulose filter is dried and exposed to high
temperature to keep the DNA in a denatured state. A la-
beled DNA probe containing a known sequence or gene
is then incubated with the nitrocellulose filter under a
variety of hybridization conditions to identify a partic-
ular fragment of DNA from the sample. Strands of the
DNA probe bind to fragments of the separated DNA that
contain similar or identical sequences and hybridize to-
gether to form double-stranded DNA. After the hybridiza-
tion reaction is complete, the solution is cooled and the
itrocellulose filter is washed to remove unbound probe
DNA and dried. The labeled DNA fragments can be vi-
sualized by autoradiography (if the probe was radioac-
tive) or by fluorescence. The DNA fragments of interest
also can be eluted from the nitrocellulose filter for further
analysis.
Polymorphisms
Approximately 25% of all human genes occur among in-
dividuals in a population in multiple allelic forms referred
to as
polymorphisms.
For example, the ABO locus, an
important factor in blood transfusions, consists of three
different alleles (alternative states of a gene) called A,
B, and O. Individuals can be homozygous or heterozy-
gous for any pair of A, B, and O alleles. The HLA family
