Page 273 - Color Atlas of Biochemistry

264 Molecular genetics
Genetic engineering in medicine
Genetic engineering procedures are becom-
ing more and more important in medicine
for diagnostic purposes (A–C). New genetic
approaches to the treatment of severe dis-
eases are still in the developmental stage
(“gene therapy,” D).
A. DNA fingerprinting
DNA fingerprinting is used to link small
amounts of biological material—e. g., traces
from thesiteof a crime—to a specificperson.
Theprocedure now usedis based on thefact
that the human genome contains non-coding
repetitive DNA sequences, the length of which
varies from individual to individual. Short
tandem repeats (STRs) thus exist in which
dinucleotides (e. g., -T-X-) are frequently re-
peated. EachSTR canoccur infive to15 differ-
ent lengths (alleles), of which one individual
possesses only one or two. When the various
allele combinations for several STRs are de-
termined after PCR amplification of the DNA
being investigated, a “genetic fingerprint” of
the individual from whom the DNA originates
is obtained. Using comparative material—e. g.,
saliva samples—definite identification is then
possible.
B. Diagnosis of sickle-cell anemia using RFLP
This example illustrates a procedure for diag-
nosing a point mutation in the β-globin gene
that leads to sickle-cell anemia (see p. 248).
The mutationinthe first exonofthe gene
destroys a cleavage site for the restriction
endonuclease MstII (see p. 258). When the
DNA of healthy and diseased individuals is
cleaved with MstII, different fragments are
produced in the region of the β-globin gene,
which can be separated by electrophoresis
and then demonstrated using specific probes
(see p. 260). In addition, heterozygotic car-
riers of the sickle-cell gene can be distin-
guished from homozygotic ones.
C. Identification of viral DNA using RT-PCR
In viral infections, it is often dif cult to deter- viral vectors to the tumor (targeting), at-
mine the species of the pathogen precisely.
RT-PCR can be used to identify RNA viruses. In
this procedure, reverse transcriptase (see
p. 404) is used to transcribe the viral RNA
into dsDNA, and then PCR is employed to
Koolman, Color Atlas of Biochemistry, 2nd edition © 2005 Thieme
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amplify a segment of this DNA with virus-
specific primers. In this way, an amplificate
with a characteristic length can be obtained
for each pathogen and identified using gel
electrophoresis as described above.
D. Gene therapy
Many diseases, such as hereditary metabolic
defects and tumors, can still not be ad-
equately treated. About 10 years ago, projects
were therefore initiated that aimed to treat
diseases of this type by transferring genes
into the affected cells (gene therapy). The
illustration combines conceivable and already
implemented approaches to gene therapy for
metabolic defects (left) and tumors (right).
None of these procedures has yet become
established in clinical practice.
If a mutation leads to failure of an enzyme
E1 (left), its substrate B will no longer be
converted into C and will accumulate. This
can lead to cell damage by B itself or by a
toxic product formed from it. Treatment
with intact E1 is not possible, as the proteins
arenot capableof passing through the cell
membrane. By contrast, it is in principle pos-
sible to introduce foreign genes into the cell
using viruses as vectors (adenoviruses or ret-
roviruses are mainly used). Their gene prod-
ucts could replace the defective E1 or convert
B into a harmless product. Another approach
uses the so-called antisense DNA (bottom
right). This consists of polynucleotides that
hybridize with the mRNA for specific cellular
proteins and thereby prevent their transla-
tion. In the case shown, the synthesis of E2
could be blocked, for example.
The main problem in chemotherapy for
tumors is the lack of tumor-specificity in the
highly toxic cytostatic agents used (see
p. 402). Attempts are therefore being made
to introduce into tumor cells genes with prod-
ucts that are only released from a precursor to
form active cytostatics once they have
reached their target (left). Other gene prod-
ucts are meant to force the cells into apoptosis
(see p. 396) or make them more susceptible
to attack by the immune system. To steer the
tempts are being made to express proteins
on the virus surface that are bound by tu-
mor-specific receptors. Fusion with a tumor-
specific promoter could also help limit the
effect of the foreign gene to the tumor cells.