there are a number of ways in which the PCR can
itself function as a diagnostic technique (Ferrari
et
al.
1996, Nollau
et al.
1997). A multibase deletion,
insertion, or expansion of trinucleotide (triplet)
repeat sequences can be detected by electrophoretic
analysis of the length of the DNA products. Using
primers that bracket the target region of DNA, the
presence of a deletion will result in the major
product of the PCR having a reduced length
compared to the major product of normal DNA.
Similarly, the presence of an insertion will lead to
the major product of the PCR being a DNA segment
with a length greater than that of the major product
of normal DNA. When performed using DNA from
an individual homozygous for the mutation, the PCR
will generate products of abnormal length. If an
individual is normal, products of normal length will
be generated and if an individual is heterozygous for
the mutation, products of both normal and abnormal
length will be generated.
Small deletions and nucleotide substitutions can
be detected by using primers that bind at the site of
the mutation. Under certain defined reaction condi-
tions, primers will not hybridize with their DNA
binding site if there is a base mismatch within the
primer and they will not support DNA synthesis if
there is a base mismatch at their 3’ residue. If the
primer is specific for the normal DNA sequence, the
PCR will generate a product from normal DNA but
not from DNA with a mutation in the primer binding
site. If the primer is specific for the mutant
sequence, normal DNA will not generate a product
but the mutant DNA will. The analysis is conducted
by performing PCR twice, once with each kind of
primer. DNA from an individual who is normal
generates products only with the primer for normal
DNA, DNA from an individual who is homozygous
for the mutation generates products only with the
primer for mutant DNA, and DNA from an individ-
ual who is heterozygous for the mutation generates
products with both primers.
Another way to detect a nucleotide substitution
is to use a primer that introduces an allele-specific
restriction enzyme cleavage site in the amplified
DNA. The primer, which binds close to the site of
the mutation, has a single base mismatch which, in
combination with the normal base at the mutation
site, produces a defined restriction enzyme cleavage
site in the newly synthesized DNA. A cleavage site
is not formed from DNA containing a mutation
because the mutant base does not generate DNA
with the proper sequence. The appropriate restric-
tion enzyme is added to the reaction mixture follow-
ing the first round of DNA amplification and normal
DNA thereby eliminated. Then, subsequent PCR
cycles will produce DNA products only if the
individual is hetero- or homozygous for the
mutation. Alternatively, the restriction enzyme
digestion step can be deferred until the PCR is
complete and the DNA products can be evaluated by
Southern blot hybridization (as discussed below).
Allele-specific oligonucleotide hybridization.
Nucleotide substitutions can be demonstrated directly
in PCR-amplified DNA by utilizing labeled oligonu-
cleotide probes that bind at the site of the mutation.
By adjusting the hybridization conditions so that only
perfectly matched duplexes are stable, signal genera-
tion occurs only if the allele match is exact. If the
probe is complementary to the normal sequence, a
signal will be generated by normal DNA. Signal
generation will occur with mutant DNA if the probe
is complementary to the mutant sequence. By
testing DNA with both types of probe, the genotype
of the individual can be ascertained. DNA from an
individual who is normal will generate a signal with
the probe for normal DNA but not with the probe
for mutant DNA. DNA from an individual who is
homozygous for a mutation will generate a signal
with the probe for the mutant DNA but not with the
probe for normal DNA. DNA from an individual
who is heterozygous will give a signal with both
probes.
Southern blot hybridization.
Southern blot hy-
bridization is based on the analysis of the lengths of
DNA fragments derived by the digestion of whole
DNA with a restriction endonuclease. The DNA
fragments are separated according to length by elec-
trophoresis in agarose gel, and transferred to a nitro-
cellulose filter by blotting. The location, and hence
the size, of the DNA fragment of interest is
indicated by hybridization of the fragment with a
radiolabeled oligonucleotide probe complementary to
Genetic Disease
10-5
Table 10.4
Severity of Genetic Disease
Gene Product
Genotype
heterozygous
homo/hemizygous
functional
normal
normal
hypofunctional
mild to moderate mild to severe
nonfunctional
mild to moderate severe
not produced mild to moderate severe
