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Vo133. No.8, August 1987
CENTRAL AFRICAN
JOURNAL OF MEDICINE
9. EvansJS, WennbergJE, McNeil BJ. The influence of
diagnostic radiography on the incidence of breast
cancer and leukaemia. N Engl J Med 1986; 315:
810-15.
10. Philpott RH, Castle WM. Cervicographs in the
management of labour in primigravidae. J Obstet
Gynaecol Br Commonw 1972; 79: 592-8.
11. Armon PI The management of patients previously
delivered by caesarean section. Cent Afr J Med 1971;
17: 170.-2.
12. Walton SM. The antenatal and intrapartum management of patients with previous caesarean section scars.
East Afr MedJ 1978; 55: 1~8.
13. Singh TKC, Barman SD. Gupta AN. Study of
vaginal delivery in patients with one previous lower
segment caesarian section. Aust NZ J Obstet
Gynaecol 1986; 26: 245.
14. Demianczuk NN, Hunter DJS, Wayne Taylor D.
Trial of labour after previous caesarean section:
Prognostic indicators of outcome. Am J Obstet
Gynaecol 1982; 142: 640-2.
15. Jagani N. Schulman H, Chandra p. Gonzolez R,
Fleischer A. The predictabilily of labour outcome
from a comparison of birth weight and X-ray pe1vimeuy.
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SPECIAL ARTICLE
already apparent in such developments as recombinant human insulin, prenatal diagnosis of
genetic disorders, and the studies of relatedness
among various strains of Human Immunodeficiency Virus (HIV). In the near future we can
expect to see applications of this technology
having an impact on many more areas of medicine
in Zimbabwe as well as elsewhere.
The basic 'double helix.' structure of DNA as
described in 1953 by Watson and CriCK is illustrated below to refresh ~our memory (Fig. 1). The
DNA is transcribed to messenger RNA which in
tum is translated into proteins according to a code
which is consistent in all life· forms.
Several developments have contributed to the
recent explosion in knowledge and practical
applications of DNA molecular biology. These
include the use of restriction endonucleases,
nucleic acid sequencing techniques, and the ability
to manipulate 'vectors' such as plasmids.
'Restriction endonucleases 'are enzymes which
have the property of cutting DNA at very specific
points. For example, an enzyme called 'Eco RI'
m~es a cut only when it encounters the nucleotide
sequence GAATIC and the endonuclease Sma I
cuts only at CCCGG. These sites provide 'landmarks' in long strands of DNA and enable a type of
mapping of an organism's genome (Fig. 2).
Advances in DNA Molecular Biology
and Their Clinical Applications: Update for Clinicians
S HOUSTON
SUMMARY
Major advances have occurred in understanding and
applications of DNA molecular biology. Selected
DNA fragments can be 'cloned', allowing manufacture of their protein products. Examples include
recombinant insulin, interferon, and vaccines using
recombinant antigens. DNA 'probes' are already
proving valuable in microbiologic diagnosis and
may have an important role in prenatal diagnosis of
inherited disease.
DISCUSSION
So much has happened in the field of molecular
biology in the last decade, that it calls for an update
on the basic science that we were taught in medical
schooL The fruits of this increased understanding are
Department o/Clinical Pharmacology,
Godfrey Huggins School 0/ Medicine.
University a/Zimbabwe.
208
Reproduced by Sabinet Gateway under licence granted by the Publisher (dated 2009).
Vol 33. No.8. Augult 1987
FIGURE I - The DNA molecule, demonstraling the specific pairing of complementary nucleotides and the
double helix structure
DNA STRUCTURE
PROTEIN
§
METHIONINE
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I
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r
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C
A
V
rr;
C . G
C
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3 5
i
...z
A
r:
GLYCINE
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T "'A
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SEAINE
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§
ISOlEVCINE ;
u
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GLYCINE
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z
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ALANINE
~
AlANINE
u
3'
0=911"'''''' C=cytosine A=adenineZT=lhymlne U=uracll
FIGURE 2 -Restriction endonuclease 'map' ofthe HN genome. Arrows indicale the sites at which virus DNA
is Clll by specijic enzymes. The 'pol' gene codesfor viralDNApolymerase (reverse transcriptase) and the 'env'
gene for the envelope proteins
RESTRICTION ENDONUCLEOSE MAP OF HUMAN IMMUNO-DEFICIENCY VIRUS
~
J.
=-
1
~~
~
1 1
1 )(1
r
~~~~~t)
11K if
pol=gene encoding reverae transcriptaae
env=gene encoding the envelope protein
the pattern of fragments produced by a given enzyme wUl be unique to thai. species. Moreover, the
fragments are of a manageable size facilitating
With a battery of these enzymes the genome of an
organism can be cUt into fragments. Since an
organism's nucleotide sequence is highly specifIC,
209
Reproduced by Sabinet Gateway under licence granted by the Publisher (dated 2009).
Vol 33. No.8, August 1987
CENTRAL AFRICAN
JOURNAL OF MEDICINI!
study (e.g. nucleotide sequencing) and manipulations such as insertion into vectors.
A vector such as a plasmid is an extrachromosomal bit of DNA ina bacterial host cell (Fig. 3). We
-~ f:lmiliar with plasmids as agents with genes
~....uing for antibiotic resistance. A plasmid can be
manipulated, for example by inserting a fragment of
foreign DNA into it The fragment will then be
replicated along with the rest of the plasmid in the
bacterial culture. That DNA fragment has now been
cloned. IT it incorporates a desirable gene, that gene
can now be produced in unlimited quantities in
cultures of host cells. Under the right conditions
these cells will use their cellular machinery to produce the product encoded by the inserted gene. Thus,
we have a mechanism for producing unlimited quantities of: (I) a desired gene or DNA sequence, and (2)
consequently, a desired gene product such as a
protein. Below are a few examples of the ways in
which these techniques can be used.
organisms. A recombinant DNA malaria vaccine,
using sporozoite antigen produced by Plasmodia
genes cloned in E. coli, is undergoing human
clinical trials. Hepatitis B vaccine produced in
yeast cells is now commercially available. It
avoids the (unfounded) concerns about the use of
the serum-derived vaccine and may reduce production costs in future. An elegant embellishment
on this process is the incorporation of cloned genes
for desired antigens into the genome of the vaccinia
(smallpox vaccine) virus. This virus then expresses
the desired antigen and stimulates specific immunity while offering the potential advantages of a
live vaccine. This approach was used in the wellpublicized French HIV vaccine currently undergoing clinical trial in Zaire.
DNA technology has advanced other areas of
biological research as well. While the sequencing
of amino acids in proteins is very difficult. the sequencing of the genes which code for them is
relatively easy. This has greatly increased our
understanding of' the structure of important biological proteins, including enzymes and the structural proteins of receptor sites.
FIGURE 3 A DNA fragment containing a desired
gene (shaded) has been inserted into an E. coli
plasmid; it has now been 'cloned'.
E.COLI CELL
Co
chromosome
Gene mutation
Genes which Can be identified and cl~ned can also
be intentionally modified structurally. One present
use of this capability is in the study of virulence
factors and pathogenic properties of micro-organisms. For example, IgA protease, an enzyme which
degrades mucosal antibody, had been postulated to
be an essential virulence factor in Neisseria gonorrhea. This theory was disproVed by observing the
unchanged virulence of strains from which the IgA
protease gene had been deleted.
In future. the ability to manipulate and alter
genes may offer the possibility of curative treatment of genetic disorders. This is a complex subject on its own which raises many ethical as well as
scientific questions, and which is beyond the scope
of this article.
plasmid containing
inserted DNA fragment
The gene for human insulin has been cloned in
bacteria allowing the production of a product which
is not only pure, but unlike pork or beef insulin.
chemically and anti genically identical to natural
human insulin. This product is already in commercial production. Using the same principle, many
difficuIt-to-obtain human biologic products such as
interferon, growth hormone, erythropoeitin, etc. can
be synthesized in unlimited quantities.
Genes encoding antigenic proteins can be cloned
in the same way and the antigens themselves produced. The resulting product is not contaminated by
potentially cross-reacting or toxic materials as is
often the case with antigen extracted from whole
DNA probes
Diagnostic DNA probes are already commercially
available for a few organisms but they are likely to
assume a far greater role in the near future. Although caution is always appropriate in proposing
210
Vol 33, No.8, August 1987
CENTRAL AFRICAN
JQL'Rl"AL OF MEDICINE'
'high tech' solutions in developing countries, some
newer probes will probably be quite modest in their
technological requirements and cost. Furthermore,
they should, at least in some circumstances, prove a
satisfactory and relatively cheap alternative to such
costly technology as viral culture,
The principle underlying the use of DNA probes is
fundamentally simple, taking advantage of two
basic characteristics of the DNA molecule: (1) The
nucleotide sequence of an organism's DNA is highly
specific to that species, (2) Single strands of DNA
which are complementary bind to each other avidly,
following base-pairing rules, to form doublestranded DNA.
The probe begins as a fragment of DNA excised
from the desired species, cloned in quantity, and then
tagged with a radioacti ve or enzyme label. The result
is a tagged molecule which will bind only to a
complementary strand of DNA, i.e. one from the
same species (Fig. 4).
DNA or RNA, or both, a probe for any organism
bacteria, fungus, virus, rickettsia, mycobacteria,
parasite, or even plasmid, can easily be designed,
Probes for mycoplasma and legionella have
reached the commercial stage. Probes have been
developed
for
Epstein
Barr
virus,
cytomegalovirus, P./alciparum, M. tuberculosis.
the plasmid of enterotoxigenic E. coli, and many
other organisms.
A somewhat similar approach can be used to
diagnose abnormalities of the genome in hereditary human disease. Two examples are the antenatal diagnosis of B. thalassemia and, using a
somewhat more complex method,ofHuntington' s
Chorea.
Probes have been used to demonstrate viral
DNA integrated into the cellular DNA of certain
tumours. For example. the finding of Hepatitis B
virus DNA incorporated into the genome of
hepatoma cells adds to the evidence that the virus
plays an aetiologic role in inducing liver cancer.
Under certain laboratory conditions, strands of
DNA of related. but not identical. organsims can be
made to hybridize. By varying these conditions. an
accurate estimate of the degree of DNA homology
or genetic relatedness between two organisms can
be worked out. These techniques have shown, for
example, the relation between mv and animal
lentiviruses. They have also been used to shed light
on the evolutionary trees of various animal species.
In summary, recent advances in DNA molecular
biology have been exciting and suggest many
potential applications in the near future.
Reproduced by Sabinet Gateway under licence granted by the Publisher (dated 2009).
FIGURE 4 Schematic illustration to show how the
DNA probe binds only to complementary DNA in the
specimen
DNA PROBES
DNA
probe
specimen 1 (negative)
radiolabel
DNA fragment
il':i:'::~
RECOMMENDED FURTHER READING
specimen 2 (positive)
probe binds to
Scientific American 1985 Oct (Molecules of Life
issue).
Nussenzweig V, Nussenzweig R. Development of
a sporozoite malaria vaccine. Am J Trop Med Jfyg
1986; 35: 67888.
Eisenstein B. Engleberg N. Applied molecular
genetics: New tools for microbiologists and
clinicians. J In/ Dis 1986; 153: 41630.
Somer SS, Sobell JL. Application of DNA-based
diagnosis to patient care: The example of
hemophilia A. Mayo Clin Proc 1987; 62: 387-404.
complementary DNA strand
The DNA probe,(e.g. one for EBV) is applied to a
filter-immobilized clinical specimen such as throat
washings or lymphocytes. If the probe remains
bound, as indicated by the persistence of the label
after washing, then EB virus must have been present
in the specimen.
The beauty of using the DNA probe approach is
that it has the potential for great specificity and
sensitivity. Since all living organisms contain either
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