Biomedical Engineering Reference
In-Depth Information
tions in kidney-transplant patients (Caballero
et al.
1997). It is worth noting that the development
of multiplex PCR technology is being facilitated by
the rapid progress in the sequencing of microbial
genomes (for an up-to-date list, see http//igweb.integ-
ratedgenomics.com/GOLD/ ), since the data gener-
ated enable species-specific genes or sequences to be
identified.
two 19-mer oligonucleotides, one of which was
complementary to the amino-terminal region of the
normal
A
) gene and the other of which
was complementary to the sickle-cell
β
-globin (
β
β
-globin gene
S
). These oligonucleotides were radiolabelled and
used to probe Southern blots. Under appropriate
conditions, the probes could distinguish the normal
and mutant alleles. The DNA from normal homo-
zygotes only hybridized with the
(
β
A
probe and DNA
from sickle-cell homozygotes only hybridized with
the
β
Comparative sequence analysis: single-
nucleotide polymorphisms (SNPs)
S
probe. DNA of heterozygotes hybridized with
both probes. These experiments, therefore, showed
that oligonucleotide hybridization probes can dis-
criminate between a fully complementary DNA and
one containing a single mismatched base. Similar
results have been obtained (Fig. 14.3) with a point
mutation in the
β
In prenatal diagnosis of genetic disorders, there is
a need to determine which alleles of a particular
locus are being carried by the fetus, i.e. is the fetus
homozygous for the normal or the deleterious allele
or is it heterozygous? In forensic DNA profiling the
requirement is to match DNA from the perpetrator
of a crime with that of a suspect. In each case, a
definitive answer could be obtained by sequencing
the relevant samples of DNA. While this is possible, it
is not practicable for mass screening. An alternative
could be to detect hybridization of specific probes.
This has been done for the detection of sickle-cell
anaemia by Conner
et al.
(1983). They synthesized
-antitrypsin gene, which is implic-
ated in pulmonary emphysema (Cox
et al.
1985).
The single-base changes that occur in the two
clinical examples just quoted are examples of single-
nucleotide polymorphisms (SNPs, pronounced
'snips'). Many such polymorphisms occur through-
out an entire genome and in humans the frequency
is about once every 1000 bases. Their distribution is
not entirely random. SNPs that alter amino acid
α
Marker
Marker
DNA
MM
MZ
ZZ
DNA
MM
MZ
ZZ
Fig. 14.3
Schematic representation
of the use of oligonucleotide probes
to detect the normal
α
1
-antitrypsin
gene (M) and its Z variant. Human
DNA obtained from normal (MM),
heterozygous (MZ) and homozygous
variant (ZZ) subjects is digested
with a restriction endonuclease,
electrophoresed and fragments
Southern blotted on to a nitrocellulose
membrane. The patterns shown were
obtained on autoradiography of the
filter following hybridization with
either the normal (M-specific) or
variant (Z-specific) probe.
M-specific probe
(C TTT CTC GTC GAT GGT CAG)
Z-specific probe
(C TTT CTT GTC GAT GGT CAG)
