Genetic “fingerprinting”

The invention: Atechnique for using the unique characteristics of
each human being’s DNA to identify individuals, establish connections
among relatives, and identify criminals.
The people behind the invention:
Alec Jeffreys (1950- ), an English geneticist
Victoria Wilson (1950- ), an English geneticist
Swee Lay Thein (1951- ), a biochemical geneticist
Microscopic Fingerprints
In 1985, Alec Jeffreys, a geneticist at the University of Leicester in
England, developed a method of deoxyribonucleic acid (DNA)
analysis that provides a visual representation of the human genetic
structure. Jeffreys’s discovery had an immediate, revolutionary impact
on problems of human identification, especially the identification
of criminals. Whereas earlier techniques, such as conventional
blood typing, provide evidence that is merely exclusionary (indicating
only whether a suspect could or could not be the perpetrator of a
crime), DNA fingerprinting provides positive identification.
For example, under favorable conditions, the technique can establish
with virtual certainty whether a given individual is a murderer
or rapist. The applications are not limited to forensic science;
DNA fingerprinting can also establish definitive proof of parenthood
(paternity or maternity), and it is invaluable in providing
markers for mapping disease-causing genes on chromosomes. In
addition, the technique is utilized by animal geneticists to establish
paternity and to detect genetic relatedness between social groups.
DNAfingerprinting (also referred to as “genetic fingerprinting”)
is a sophisticated technique that must be executed carefully to produce
valid results. The technical difficulties arise partly from the
complex nature of DNA. DNA, the genetic material responsible for
heredity in all higher forms of life, is an enormously long, doublestranded
molecule composed of four different units called “bases.”
The bases on one strand of DNApair with complementary bases on the other strand. A human being contains twenty-three pairs of
chromosomes; one member of each chromosome pair is inherited
fromthe mother, the other fromthe father. The order, or sequence, of
bases forms the genetic message, which is called the “genome.” Scientists
did not know the sequence of bases in any sizable stretch of
DNA prior to the 1970’s because they lacked the molecular tools to
split DNA into fragments that could be analyzed. This situation
changed with the advent of biotechnology in the mid-1970’s.
The door toDNAanalysis was opened with the discovery of bacterial
enzymes called “DNA restriction enzymes.” A restriction enzyme
binds to DNA whenever it finds a specific short sequence of
base pairs (analogous to a code word), and it splits the DNAat a defined
site within that sequence. A single enzyme finds millions of
cutting sites in human DNA, and the resulting fragments range in
size from tens of base pairs to hundreds or thousands. The fragments
are exposed to a radioactive DNA probe, which can bind to
specific complementary DNA sequences in the fragments. X-ray
film detects the radioactive pattern. The developed film, called an
“autoradiograph,” shows a pattern of DNA fragments, which is
similar to a bar code and can be compared with patterns from
known subjects.
The Presence of Minisatellites
The uniqueness of a DNA fingerprint depends on the fact that,
with the exception of identical twins, no two human beings have
identical DNA sequences. Of the three billion base pairs in human
DNA, many will differ from one person to another.
In 1985, Jeffreys and his coworkers, Victoria Wilson at the University
of Leicester and Swee Lay Thein at the John Radcliffe Hospital
in Oxford, discovered a way to produce a DNA fingerprint.
Jeffreys had found previously that human DNA contains many repeated
minisequences called “minisatellites.” Minisatellites consist
of sequences of base pairs repeated in tandem, and the number of
repeated units varies widely from one individual to another. Every
person, with the exception of identical twins, has a different number
of tandem repeats and, hence, different lengths of minisatellite
DNA. By using two labeled DNA probes to detect two different minisatellite sequences, Jeffreys obtained a unique fragment band
pattern that was completely specific for an individual.
The power of the technique derives from the law of chance,
which indicates that the probability (chance) that two or more unrelated
events will occur simultaneously is calculated as the multiplication
product of the two separate probabilities. As Jeffreys discovered,
the likelihood of two unrelated people having completely
identical DNAfingerprints is extremely small—less than one in ten
trillion. Given the population of the world, it is clear that the technique
can distinguish any one person from everyone else. Jeffreys
called his band patterns “DNAfingerprints” because of their ability
to individualize. As he stated in his landmark research paper, published
in the English scientific journal Nature in 1985, probes to
minisatellite regions of human DNA produce “DNA ‘fingerprints’
which are completely specific to an individual (or to his or her identical
twin) and can be applied directly to problems of human identification,
including parenthood testing.”
Consequences
In addition to being used in human identification, DNA fingerprinting
has found applications in medical genetics. In the search
for a cause, a diagnostic test for, and ultimately the treatment of an
inherited disease, it is necessary to locate the defective gene on a human
chromosome. Gene location is accomplished by a technique
called “linkage analysis,” in which geneticists use marker sections
of DNA as reference points to pinpoint the position of a defective
gene on a chromosome. The minisatellite DNA probes developed
by Jeffreys provide a potent and valuable set of markers that are of
great value in locating disease-causing genes. Soon after its discovery,
DNA fingerprinting was used to locate the defective genes responsible
for several diseases, including fetal hemoglobin abnormality
and Huntington’s disease.
Genetic fingerprinting also has had a major impact on genetic
studies of higher animals. BecauseDNAsequences are conserved in
evolution, humans and other vertebrates have many sequences in
common. This commonality enabled Jeffreys to use his probes to
human minisatellites to bind to the DNA of many different vertebrates, ranging from mammals to birds, reptiles, amphibians, and
fish; this made it possible for him to produce DNA fingerprints of
these vertebrates. In addition, the technique has been used to discern
the mating behavior of birds, to determine paternity in zoo primates,
and to detect inbreeding in imperiled wildlife. DNA fingerprinting
can also be applied to animal breeding problems, such as
the identification of stolen animals, the verification of semen samples
for artificial insemination, and the determination of pedigree.
The technique is not foolproof, however, and results may be far
from ideal. Especially in the area of forensic science, there was a
rush to use the tremendous power of DNA fingerprinting to identify
a purported murderer or rapist, and the need for scientific standards
was often neglected. Some problems arose because forensic
DNA fingerprinting in the United States is generally conducted in
private, unregulated laboratories. In the absence of rigorous scientific
controls, the DNA fingerprint bands of two completely unknown
samples cannot be matched precisely, and the results may be
unreliable.

Antibacterial drugs

Mechanisms of genetic resistance to antimicrobial agents:

Bacteria have developed, or will develop, genetic resistance to all known antimicrobial agents that are now in the marketplace. The five main mechanisms that bacteria use to resist antibacterial drugs are shown in the figure.
a | The site of action (enzyme, ribosome or cell-wall precursor) can be altered. For example, acquiring a plasmid or transposon that codes for a resistant dihydrofolate reductase confers trimethoprim resistance to bacteria52.
b | The inhibited steps can be by-passed.
c | Bacteria can reduce the intracellular concentration of the antimicrobial agent, either by reducing membrane permeability, for example, as shown by Pseudomonas aeruginosa53, or by active efflux of the agent54.
d | They can inactivate the drug. For example, some bacteria produce beta-lactamase, which destroys the penicillin beta-lactam ring50, 51 .
e | The target enzyme can be overproduced by the bacteria.



The invention:

Sulfonamides and other drugs that have proved effective
in combating many previously untreatable bacterial diseases.

The people behind the invention:

Gerhard Domagk (1895-1964), a German physician who was
awarded the 1939 Nobel Prize in Physiology or Medicine
Paul Ehrlich (1854-1915), a German chemist and bacteriologist
who was the cowinner of the 1908 Nobel Prize in Physiology
or Medicine.

The Search for Magic Bullets

Although quinine had been used to treat malaria long before the
twentieth century, Paul Ehrlich, who discovered a large number of
useful drugs, is usually considered the father of modern chemotherapy.
Ehrlich was familiar with the technique of using dyes to stain
microorganisms in order to make them visible under a microscope,
and he suspected that some of these dyes might be used to poison
the microorganisms responsible for certain diseases without hurting
the patient. Ehrlich thus began to search for dyes that could act
as “magic bullets” that would destroy microorganisms and cure
diseases. From 1906 to 1910, Ehrlich tested numerous compounds
that had been developed by the German dye industry. He eventually
found that a number of complex trypan dyes would inhibit the
protozoans that caused African sleeping sickness.
Ehrlich and his coworkers also synthesized hundreds of organic
compounds that contained arsenic. In 1910, he found that one of
these compounds, salvarsan, was useful in curing syphilis, a sexually
transmitted disease caused by the bacterium Treponema. This
was an important discovery, because syphilis killed thousands of
people each year. Salvarsan, however, was often toxic to patients,
because it had to be taken in large doses for as long as two years to
effect a cure. Ehrlich thus searched for and found a less toxic arsenic
compound, neosalvarsan, which replaced salvarsan in 1912.

In 1915, tartar emetic (a compound containing the metal antimony)
was found to be useful in treating kala-azar, which was
caused by a protozoan. Kala-azar affected millions of people in Africa,
India, and Asia, causing much suffering and many deaths each
year. Two years later, it was discovered that injection of tartar emetic
into the blood of persons suffering from bilharziasis killed the
flatworms infecting the bladder, liver, and spleen. In 1920, suramin,
a colorless compound developed from trypan red, was introduced
to treat African sleeping sickness. It was much less toxic to the patient
than any of the drugs Ehrlich had developed, and a single dose
would give protection for more than a month. From the dye methylene
blue, chemists made mepacrine, a drug that was effective
against the protozoans that cause malaria. This chemical was introduced
in 1933 and used duringWorldWar II; its principal drawback
was that it could cause a patient’s skin to become yellow.

Well Worth the Effort

Gerhard Domagk had been trained in medicine, but he turned to
research in an attempt to discover chemicals that would inhibit or
kill microorganisms. In 1927, he became director of experimental
pathology and bacteriology at the Elberfeld laboratories of the German
chemical firm I. G. Farbenindustrie. Ehrlich’s discovery that
trypan dyes selectively poisoned microorganisms suggested to Domagk
that he look for antimicrobials in a new group of chemicals
known as azo dyes. A number of these dyes were synthesized
from sulfonamides and purified by Fritz Mietzsch and Josef Klarer.
Domagk found that many of these dyes protected mice infected
with the bacteria Streptococcus pyogenes. In 1932, he discovered that
one of these dyes was much more effective than any tested previously.
This red azo dye containing a sulfonamide was named prontosil
rubrum.
From 1932 to 1935, Domagk began a rigorous testing program to
determine the effectiveness and dangers of prontosil use at different
doses in animals. Since all chemicals injected into animals or humans
are potentially dangerous, Domagk determined the doses that
harmed or killed. In addition, he worked out the lowest doses that
would eliminate the pathogen. The firm supplied samples of the drug to physicians to carry out clinical trials on humans. (Animal
experimentation can give only an indication of which chemicals
might be useful in humans and which doses are required.)
Domagk thus learned which doses were effective and safe. This
knowledge saved his daughter’s life. One day while knitting, Domagk’s
daughter punctured her finger with a needle and was infected
with a virulent bacteria, which quickly multiplied and spread
from the wound into neighboring tissues. In an attempt to alleviate
the swelling, the infected area was lanced and allowed to drain, but
this did not stop the infection from spreading. The child became
critically ill with developing septicemia, or blood poisoning.
In those days, more than 75 percent of those who acquired blood
infections died. Domagk realized that the chances for his daughter’s
survival were poor. In desperation, he obtained some of the powdered
prontosil that had worked so well on infected animals. He extrapolated
from his animal experiments how much to give his
daughter so that the bacteria would be killed but his daughter
would not be poisoned. Within hours of the first treatment, her fever
dropped, and she recovered completely after repeated doses of
prontosil.

Impact

Directly and indirectly, Ehrlich’s and Domagk’s work served to
usher in a new medical age. Prior to the discovery that prontosil
could be use to treat bacterial infection and the subsequent development
of a series of sulfonamides, or “sulfa drugs,” there was no
chemical defense against this type of disease; as a result, illnesses
such as streptococcal infection, gonorrhea, and pneumonia held terrors
of which they have largely been shorn.Asmall injury could easily
lead to death.
By following the clues presented by the synthetic sulfa drugs and
how they worked to destroy bacteria, other scientists were able to
develop an even more powerful type of drug, the antibiotic. When
the American bacteriologist Rene Dubos discovered that natural organisms
could also be used to fight bacteria, interest was renewed in
an earlier discovery by the Scottish bacteriologist Sir Alexander: the
development of penicillin.
Antibiotics such as penicillin and streptomycin have become
some of the most important tools in fighting disease. Antibiotics
have replaced sulfa drugs for most uses, in part because they cause
fewer side effects, but sulfa drugs are still used for a handful of purposes.
Together, sulfonamides and antibiotics have offered the possibility
of a cure to millions of people who previously would have
had little chance of survival.