Today, Sherlock Holmes would be a molecular biologist. For each of us, save
identical twins, carries a unique genetic pattern in our DNA. It would be a
case of 鈥淓lementary, my dear Watson!鈥
IN July 1977, Monica Lemos de Lavalle, by then eight months鈥 pregnant, was
abducted by the security forces in the province of Buenos Aires, Argentina,
with her husband and their 14-month-old daughter, Maria. Five days later,
Maria was abandoned by her abductors near the house of her maternal
grandmother. The fate of the young couple, and the baby Monica carried,
remained unknown. Then a man just released from a secret concentration camp in
Argentina told the grandmother that her daughter Monica had also been
imprisoned there.
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In 1985, authorities from the new democratic government questioned a former
policewoman who had worked at the camp, and had a daughter she claimed as her
own. Two years later, genetic tests carried out by the new government鈥檚
Genetic Data Bank showed with a 99.98 per cent certainty that the girl was in
fact the daughter of Monica Lemos de Lavalle. The child is now reunited with
her sister and grandmother, although her parents are still missing, presumed
dead.
These new genetic tests, known as genetic fingerprinting, are now helping
to reunite with their grandparents many children who 鈥渄isappeared鈥 during the
period of military rule in Argentina (1976-1983). Because the grandparents of
the kidnapped children are aging, and many of the children are still missing,
the Argentinian government agreed to establish a genetic data bank in May
1987. The bank stores blood samples from the relatives of missing children,
and carries out the genetic fingerprinting tests that can identify their lost
children.
Genetic fingerprinting was developed by Alec Jeffreys at the University of
Leicester in 1984. He was studying the gene for myoglobin, a protein that
stores oxygen in muscles. He found that part of the gene did not carry
instructions for the manufacture of myoglobin. Instead, this bit of DNA
consisted of an unusual sequence of bases, repeated several times. These
repeated sequences, called minisatellites, seem to be useless but harmless
bits of DNA. Yet Jeffreys realised they might prove useful to geneticists. He
thought that these variable, short sequences, each about 10 to 15 base-pairs
long, could act as genetic markers for the myoglobin gene 鈥 helpful in
tracking down its location on a particular chromosome.
To produce large quantities of the markers, Jeffreys isolated two
minisatellites and introduced them into bacteria. He then extracted the
multiplied markers from the bacteria and labelled them with radioactive
chemicals. Now he had genetic probes that would bind to and so reveal the
presence of minisatellites in any DNA sample.
But another surprise was in store. Jeffreys discovered that these
minisatellite sequences were repeated many times in different parts of the
DNA, not just in the myoglobin gene. Stranger still, the pattern of these
repeats turned out to be different in each individual. Everyone has these
repeated minisatellite regions in their DNA, but the number of repeats is
unique to every individual. Moreover, each of us inherits these repeats from
our parents, half of them from our mother and half from our father. Only
identical twins end up with the same numbers of minisatellite sequences.
A DNA probe (or genetic marker) is a synthetic length of DNA, made in the
laboratory. The DNA fingerprinting process depends on DNA probes that are made
up of repeated sequences of bases. Geneticists splice the extended repeats
into the DNA of a circular piece of DNA known as a plasmid and introduce it
into bacteria 鈥 a procedure known as cloning. The bacteria then treat the
foreign plasmid as their own and make more copies of it as they reproduce. To
make a batch of DNA probe, researchers grow the bacteria in the laboratory,
isolate the plasmids and extract the repeated sequences of DNA. It is then
easy to attach a radioactive label to the probes, by replacing phosphate
molecules with a radioactive isotope of phosphate.
Making a DNA fingerprint
Patterns in the gel
TO MAKE a genetic fingerprint, you need a sample of any tissue that
contains cells 鈥 blood, saliva, hair roots or semen will do. The cells contain
DNA, housed in the chromosomes within a cell鈥檚 nucleus. Biologists extract the
DNA from the sample and cut it at specific points using so-called restriction
enzymes. The fragments of DNA, placed on an agarose gel, are separated by
running an electric current through the gel, a process known as
electrophoresis. The pieces of DNA have a negative charge, so when a
positively charged electrode is placed at the other end of the gel, the
charged fragments of DNA travel towards it through the gel. The shorter,
lighter fragments travel more quickly through the gel, while the longer,
heavier fragments move more slowly. So this tends to separate the fragments
according to their size.
The pattern on the gel, invisible at this stage, is covered by a nylon
membrane and a layer of paper towels. The towels draw the DNA fragments
upwards into the nylon membrane by capillary action. Radioactive DNA probes
are now applied to the membrane, and these bind to any complementary
minisatellite sequences. Excess, unattached probe is simply washed away.
To make the pattern of bound minisatellite fragments visible, researchers
now put an X-ray film next to the membrane. The places where the radioactive
DNA probe has bound to the DNA fragments causes a visible fogging on the film.
This creates a pattern of bands, or a DNA profile, commonly called a DNA
fingerprint.
Bar codes for biologists
Contaminant problems
THE FINAL DNA fingerprint is a pattern on X-ray film of light and dark
bands 鈥 darker bands contain more DNA fragments. It looks a bit like the bar
codes now found on retail goods. Researchers can describe the profile as a
series of numbers, each referring to the length of the DNA fragment 鈥
calculated by measuring how far the fragment has travelled through the gel
compared with marker bands of DNA of known length.
Although these techniques are now standard practice in molecular biology
laboratories, great care must be taken in carrying out genetic fingerprinting
tests. Forensic samples of DNA are rarely pure: typically, bloodstains on
clothing or furnishing fabrics will be contaminated with all sorts of other
material. In rape cases, the vaginal or anal swab used to obtain a sample of
the attacker鈥檚 semen also contains cells from the victim, so the victim鈥檚 DNA
must also be analysed alongside the sample from the alleged attacker. DNA from
fungi and bacteria are also invariably present, and show up in the
fingerprint. DNA decays rapidly especially in warm or damp conditions. If the
DNA has decayed, some of the DNA sites attacked by restriction enzyme may be
lost, and thus result in too few or too many DNA fragments.
Any contaminants, such as dyes from blue denim jeans, can combine with the
restriction enzymes, causing them to cut in the wrong places, so that there are
too few or too many pieces of DNA. When contaminated fragments of DNA are
sorted in the gel, they may be mixed with ions that affect their charge, so
that they travel different distances through the gel and shift the bands to an
unpredictable extent. Proteins from the environment can have the same effect,
by attaching to the DNA fragments and weighing them down.
If the DNA is contaminated, even DNA samples from the same person can give
DNA fingerprints where the ends do not align, although their relative
positions remain the same 鈥 a problem known as band shift. In the probing
stage at the end of the process, probes can bind to the contaminating DNA
rather than the human sample, causing spurious bands, or the contaminant may
interfere with the probe, so it does not bind as expected, leading to missing
bands.
Assumptions and doubts
Problems with relatives
GENETIC fingerprinting also relies on the assumption that people within a
country marry and have children at random and therefore that the bands present
in a genetic fingerprint are distributed randomly in a population. Some
studies in the US cast doubt on this assumption. In most Western countries,
there are many smaller ethnic and religious groups where certain genetic
characteristics may be more common.
Members of such groups are likely to marry within their community, and so
tend to be somewhat more closely related genetically than the population as a
whole. This modest 鈥渋nbreeding鈥 increases the chances that a child will
inherit the same repeated sequence from both parents 鈥 and so be homozygous
for that sequence. Homozygotes carry one, rather than two, bands for a given
probe because they have received the same DNA sequence from each parent. Their
genetic fingerprint will show one dark band rather than two lighter ones. This
can bias calculations based on the assumption that everyone will have two
bands.
Despite these problems, genetic fingerprinting has been widely used in the
British courts to secure convictions. But responses to the technique in the US
courts have been more guarded. In some cases courts have rejected DNA
evidence. The difficulty is to ensure that rigorous standards are always
upheld. 鈥淏lind鈥 trials have been carried out in the US, with different
commercial laboratories running DNA tests on the same samples. In these
trials, some laboratories failed to identify mixed DNA as coming from two
individuals, or found false positives (declaring a match between samples which
in fact came from different individuals). European forensic laboratories are
now trying to agree on standard techniques for producing genetic fingerprints,
in an attempt to overcome some of the criticisms raised against the technique
in the US. Standardising the technique means agreeing on which restriction
enzymes and probes to use, as well as running a known set of controls for
comparison and checking that probes are uncontaminated. Most experts agree
that the technique still shows as much promise as ever, provided that proper
precautions are taken to obtain uncontaminated samples and avoid sloppy
laboratory practices.
DNA forensics
Identification of rapists
A VAGINAL swab taken from the victim can be used to obtain a DNA
fingerprint. The swab holds DNA from both the victim and the rapist. The
complex fingerprint that results must then be compared with the victim鈥檚 DNA
fingerprint, made from a sample of the victim鈥檚 blood cells. Any bands derived
from the vaginal swab DNA not present in the victim鈥檚 blood cell sample must
have come from the rapist. This technique means that not only can the rapist
be identified with a high degree of certainty, but also wrongly-accused
suspects can be proven innocent.
Other criminals, such as murderers and burglars, can also be identified
provided that a biological specimen, such as a minute drop of blood or a few
hair roots, has been left at the scene of the crime. A genetic fingerprint can
be produced from as little as 50 microlitres of blood, 5 microlitres of semen
or 10 hair roots. Mouth swabs, fetal material and muscle tissue from dead
bodies are also suitable sources of DNA.
DNA fingerprinting can also help forensic scientists to identify corpses
that are decomposed, or unidentifiable because only part of a body, such as an
arm, has been found. Comparison of the DNA fingerprint from the body with
those of living relatives, such as parents, brothers and sisters, can lead to
a positive identification. Jeffreys has extracted DNA from bones supposed to
be those of Joseph Mengele, the Nazi war criminal, and is now trying to obtain
blood samples from living relatives to determine whether the remains really
are Mengele鈥檚.
Because a child inherits half its DNA from its mother and half from its
father, DNA fingerprints can be used in disputes about who the father is. The
bands in a child鈥檚 DNA fingerprint that do not match its mother鈥檚 must come
from the child鈥檚 father. This has proved invaluable in immigration disputes. A
Home Office-sponsored pilot study commissioned from Cellmark Diagnostics and
Jeffreys in 1985 examined 36 families wishing to enter Britain to join their
relatives. The study found that nearly 90 per cent of the cases were genuine 鈥
although half of the applications had previously been rejected due to lack of
sufficient evidence to convince the immigration authorities that the people
really were related.
Researchers find DNA fingerprinting useful in studies of wild animals too.
Only about 20 Californian condors remain in the US, split between San Diego
Zoo and Los Angeles Zoo. The condors have now been 鈥渇ingerprinted鈥 so that
zookeepers can establish relatively unrelated breeding pairs to encourage
variation in the offspring. San Diego Zoo has a similar programme with its
Galapagos tortoises.
Our ultimate identity card
In sickness and in health
THE TECHNIQUE can also help doctors to monitor bone marrow transplants.
Leukaemia is a cancer of the bone marrow and the diseased marrow must be
removed. But bone marrow makes new blood cells, so the sufferer will die
without a transplant of healthy marrow. Doctors can quickly tell whether the
transplant has succeeded by taking DNA fingerprints. If the transplant has
worked, a fingerprint from the patient鈥檚 blood shows the donor鈥檚 bands. But if
the cancerous bone marrow has not been properly destroyed, then the cancerous
cells multiply rapidly and the patient鈥檚 own bands predominate.
Geneticists have also used the technique of genetic fingerprinting to
identify identical twins. If twins are truly identical, formed by the division
of a single fertilised egg, they have identical genetic fingerprints. The
technique can confirm that twins who have been separated since early childhood
and brought up in different environments really are genetically identical.
This information is important because the study of twins can shed light on
many diseases that have both a genetic and environmental component, such as
cancer, depression, diabetes, Alzheimer鈥檚 disease and schizophrenia.
Geneticists studying inherited diseases such as cystic fibrosis are now
using genetic fingerprinting to analyse blood samples taken from members of a
family with an affected child. Such studies can help researchers to track down
the gene responsible, and discover where it is located on a particular
chromosome. Probes that bind to DNA in many places can act as genetic markers
to locate a region inherited in the same way as the disease. This shows that
this DNA region is physically very close on the chromosome to the particular
disease gene. Ellen Solomon and her team of researchers at the Imperial Cancer
Research Fund have used the technique to pinpoint the gene for familial
polyposis coli, a type of colon cancer that affects people in their early 20s,
and which is now known to be located on chromosome 5.
Many more applications for DNA fingerprinting will no doubt be developed.
Indeed, one day hospitals may take DNA fingerprints of all newborn babies and
store the information on computer, turning each person鈥檚 genetic material into
the ultimate, lifelong identity card.
Probing DNA
TWO sorts of probes are used to produce most genetic fingerprints. One
sort, the multi-locus probes, are a short length of radioactive double-
stranded DNA. These probes bind onto the DNA in many places and produce a DNA
fingerprint with many bands for comparison 鈥 especially important in paternity
testing. In unrelated people, only one in four bands (0.25) match another
person鈥檚 DNA fingerprint. So if there are, say, 10 bands in a DNA fingerprint,
the chance match of these is expected once in 0.210, or
approximately once in a million individuals. When many bands are analysed,
paternity is established with virtual certainty. Multi-locus probes are also
favoured in forensic investigations when the sample is plentiful and in good
condition.
Single-locus probes are copies of a single long length of DNA (often part
of a chromosome) which bind to the DNA at only one point. This type of probe
produces only two bands in any individual, one from the mother and one from
the father. Less DNA is needed than when using multi-locus probes because the
large single-locus probes give such a strong signal, and so are best when the
sample is tiny. Even partially decomposed DNA often shows a clear profile
pattern with single-locus probes. They also allow mixtures of biological
samples to be analysed. Because each individual usually produces two bands, a
vaginal swab from a rape victim yielding eight bands suggests that the victim
was raped by three men.
Forensic investigations commonly use three or four different single-locus
probes on a single sample to produce DNA prints consisting of six to eight
bands. Using more than one single-locus probe improves the valdiity of the
test.
Restriction enzymes
MANY bacteria make restriction enzymes to protect themselves from invading
foreign DNA molecules such as viruses. The restriction enzymes attack the
invading DNA by chopping it up. Each kind of enzyme (there are hundreds)
recognises a particular, and different, sequence of between four and six bases
and cuts the DNA there. Many restriction enzymes have been purified from
various species of bacteria, and more than 100 are commercially available. For
example, Eco RI comes from Escherichia coli and Hind III from Haemophilus
influenzae.
The polymerase chain reaction
SCIENTISTS at the Cetus Corporation in the US in 1985 invented the
polymerase chain reaction (PCR). It is an ingenious technique for copying
minute amounts of DNA many times over and has already revolutionised genetic
testing. The procedure can 鈥渁mplify鈥, or make more copies of, a tiny bit of
DNA 鈥 so that there is enough for tests such as genetic fingerprinting even if
you start out with only the tiny amount of DNA contained in a single cell.
The technique works by exploiting the ability of an enzyme, DNA polymerase,
to make copies of DNA. First, researchers heat the DNA, so that the double
helix unwinds. Short sequences of DNA, called primers, are then attached to
each end of each single strand of the target piece of DNA. These primers tell
the enzyme where to start making copies. Next, researchers add the enzyme DNA
polymerase, together with a mixture of free bases. The enzyme directs the
manufacture of a complementary sequence of DNA, making two complete double
strands of DNA, both identical to the original piece of DNA. This cycle is
repeated many times over, each doubling the amount of DNA in the sample.
This technique has not yet been widely used forensically, partly because
PCR is particularly sensitive to contamination and partly because it involves
鈥渕anufactured鈥 DNA which is not readily accepted as evidence in court. But it
is a useful technique in all sorts of research. Even archaeologists have
exploited it. For instance, Erika Hagelberg at the John Radliffe Hospital in
Oxford, working with colleagues from the University of Oxford鈥檚 archaeology
unit, has extracted mitochondrial DNA from bones of English Civil War
skeletons, and amplified it using PCR 鈥 giving clues to our genetic history.
One interesting forensic application of PCR arose when the remains of a
body wrapped in a carpet were discovered at a house in Cardiff in 1989. Facial
reconstruction led the police to guess that the body was that of Karen Price,
a teenager who had disappeared in 1981, but no one could be sure. Erika
Hagelberg extracted a tiny amount of DNA from one of the bones, and sent it
for analysis to Alec Jeffreys at Leicester.
The sample was heavily contaminated with microbial DNA and decayed, but by
using PCR the researchers amplified six minisatellite regions. By comparing
these sequences with DNA from Karen Price鈥檚 parents, the evidence was strong
that the body was indeed that of Karen Price. In February 1991 this was
accepted by Cardiff Crown Court, the first time PCR has been accepted as
evidence in a British court.
Further reading
鈥淭he oldest DNA in the world鈥, Julie Johnson, New Scientist, 11 May 1991.
鈥淚s DNA fingerprinting ready for the courts?鈥, William C. Thompson and Simon
Ford, New Scientist, 31 March 1990.