انت هنا الان : شبكة جامعة بابل > موقع الكلية > نظام التعليم الالكتروني > مشاهدة المحاضرة

lec9

Share |
الكلية كلية العلوم للبنات     القسم قسم علوم الحياة     المرحلة 7
أستاذ المادة اسراء عدنان ابراهيم البغدادي       20/06/2017 08:30:14
DNA pro?ling
Given the size of the human genome, and our knowledge of genome
structure, it is relatively easy to calculate that each person’s genome is
unique, the only exceptions being monozygotic twins (twins derived
from a single fertilised ovum). This provides the opportunity to use
DNA pro?ling techniques exploit
the fact that each genome is
unique (apart from the genomes
of monozygotic twins).
the genome as a unique identi?er, if suitable techniques are avail-
able to generate robust and unambiguous results. The original tech-
nique was called DNA fingerprinting, but with improved technology
the range of tests that can be carried out has increased, and today
the more general term DNA profiling is preferred. The technique has
found many applications in both criminal cases and in disputes over
whether people are related or not (paternity disputes and immigra-
tion cases are the most common). The basis of all the techniques is
that a sample of DNA from a suspect (or person in a paternity or immi-
gration dispute) can be matched with that of the reference sample
(from the victim of a crime, or a relative in a civil case). In scene-
of-crime investigations, the technique can be limited by the small
amount of DNA available in forensic samples. Modern techniques use
the PCR to amplify and detect minute samples of DNA from blood-
stains, body ?uids, skin fragments, or hair roots.
12.4.1 The history of ‘genetic ?ngerprinting’
The original DNA ?ngerprinting technique was devised in 1985 by
Alec Jeffreys, who realised that the work he was doing on sequences
within the myoglobin gene could have wider implications. The
method is based on the fact that there are highly variable regions of
the genome that are speci?c to each individual. These are minisatel-
lite regions, which have a variable number of short repeated-sequence
elements known as variable number tandem repeats (VNTRs, see
Chapter 10 and Fig. 10.10). Within the VNTR there are core sequence
motifs that can be identi?ed in other polymorphic VNTR loci, and also
sequences that are restricted to the particular VNTR. The arrangement
of the VNTR sequences, and the choice of a suitable probe sequence,
are the key elements that enable a unique ‘genetic ?ngerprint’ to be
produced.
The ?rst requirement is to isolate DNA and prepare restriction
fragments for electrophoresis. As shown in Fig. 10.10, if an enzyme
is used that does not cut the core sequence, but cuts frequently out-
side it, then the VNTR is effectively isolated. For human DNA theMEDICAL AND FORENSIC APPLICATIONS OF GENE MANIPULATION 249
(a)
(b)
VNTR
Digest with fI Hin
electrophoresis
VNTR fragments
(c)
(d)
MLP
SLP
Fig. 12.8 Genetic ?ngerprinting of minisatellite DNA sequences. (a) A chromosome
pair, with one minisatellite (VNTR) locus highlighted. In this case the locus is
heterozygous for VNTR length. Cutting with HinfI effectively isolates the VNTR. (b) The
VNTR fragments produced (from many loci) are separated by electrophoresis and
blotted. Challenging with a multi-locus probe (MLP) produces the ‘bar code’ pattern
shown in (c). If a single-locus probe (SLP) is used, the two alleles of the speci?c VNTR
are identi?ed as shown in (d).
enzyme HinfI is often used. By using a probe that hybridises to the
core sequence, and carrying out the hybridisation under low strin-
gency, polymorphic loci that bind the probe can be identi?ed. The
The original method of
generating a DNA pro?le
(sometimes called DNA
?ngerprinting) produces results
in the now familiar ‘bar code’
format.
probe in this case is known as a multi-locus probe, as it binds to
multiple sites. This generates a pattern of bands that is unique -- the
‘genetic ?ngerprint’. If probes with sequences that are speci?c for
a particular VNTR are used (single-locus probes), a more restricted
?ngerprint is produced, as there will be two alleles of the sequence
in each individual, one maternally derived and one paternally derived.
An overview of the basis of the technique is shown in Fig. 12.8.
In forensic analysis, the original DNA pro?ling technique has now
been largely replaced by a PCR-based method that ampli?es parts
of the DNA known as short tandem repeats (STRs, also known as
microsatellites). These are repeats of 2, 3, 4, or 5 base pairs. A major250 GENETIC ENGINEERING IN ACTION
advantage over minisatellite (VNTR) repeats is that STRs are found
throughout the genome; thus, better coverage is achieved than with
minisatellites. The PCR overcomes any problems associated with the
tiny amounts of sample that are often found at the crime scene. The
reaction is set up to amplify the loci involved -- usually 3 or 4 are
suf?cient if the loci are selected carefully to optimise the information
generated. By using ?uorescent labels and automated DNA detection
equipment (similar to the genome sequencing equipment shown in
Fig. 10.8) a DNA pro?le can be generated quickly and accurately.
12.4.2 DNA pro?ling and the law
The use of DNA pro?ling is now accepted as an important way of gen-
erating evidence in legal cases. In addition to the science itself, which
may involve multi-locus or single-locus probes, or (more usually) STR
ampli?cation, there are several factors that must be considered if the
evidence is to be sound. The techniques must be reliable and must be
accessible to trained technical staff, who must be aware of the poten-
tial problems with the use of DNA pro?ling. To ensure that results
from DNA pro?le analysis are admissible as evidence in legal cases,
rigorous quality control measures must be in place. These include
accurate recording of the samples as they arrive at the laboratory,
and careful cross-checking of the procedures to make sure that the
test is carried out properly and that the samples do not get mixed up.
To be useful in a legal context,
DNA pro?ling must be managed
and regulated within an agreed
framework so that the public can
have con?dence in the
procedure. If PCR ampli?cation is used as part of the procedure, great care must
be taken to ensure that no trace of DNA contamination is present. A
smear of the operator’s sweat can often be enough to ruin a test, so
strict operating procedures must be observed and laboratories must
be inspected and authorised to conduct the tests. This is essential if
public con?dence in the technique is to be maintained.
As well as the detail of laboratory protocols, and the operational
management of forensic laboratories, an important part of ensuring
the acceptance of DNA evidence is the scienti?c and legal framework
within which the procedures are carried out. The use of STR-based
methods has resulted in the setting of accepted standard protocols
and gene loci or target sequences. In Europe this is overseen by the
European Network of Forensic Science Institutes, and in the USA by
the American FBI’s Combined DNA Index System.
An important consideration in legal cases is the likelihood of
matching DNA pro?les being generated by chance from two different
individuals. This is obviously critical in cases where legal decisions
are made on the strength of DNA ?ngerprint evidence, and perhaps
custodial sentences passed. Although there is no dispute about the
fact that we all have unique genomes, DNA pro?ling of course can
only examine a small part of the genome. Thus, the odds of a chance
match need to be calculated. The more bands present in a DNA pro-
?le, the less likely a non-related match will be found. The odds against
a chance match for varying numbers of bands in a DNA pro?le are
shown in Table 12.5. It is generally accepted that an approved DNA
pro?ling agency, working under agreed conditions with standardisedMEDICAL AND FORENSIC APPLICATIONS OF GENE MANIPULATION 251
Table 12.5. The odds against chance
matches in a DNA ?ngerprint
Number of bands
in ?ngerprint
Odds against a
chance match
4 250: 1
6 4 000: 1
8 65 000: 1
10 1 million: 1
12 17 million: 1
14 268 million: 1
16 4 300 million: 1
18 68 000 million: 1
20 1 million million: 1
Note: The more bands present in a DNA fingerprint,
the less likely it is that any match is due to chance.
However, allele frequencies for different genes may
have to be taken into account. Allele frequencies can
vary in different populations, and again this may
be important in a legal situation. Generally, prob-
lems can be avoided by taking all known factors into
account and assessing the risk of a chance match by
taking the highest estimate.
Source: Data courtesy of Cellmark Diagnostics. Repro-
duced with permission.
protocols, will generate results that are valid and reliable. As case law
has developed in forensic applications of DNA analysis, acceptance
has increased and the methods are now seen as robust. In addition to
dealing with new cases, DNA evidence is also being used to revisit so-
called ‘cold cases’ and in many of these to either con?rm or overturn
original convictions.
An example of the use of multi-locus DNA pro?ling in a forensic
case is shown in Fig. 12.9. In this example, blood from the victim
is the reference sample. Samples from seven suspects were obtained
and treated along with the sample from the victim. By matching the
band patterns it is clear that suspect 5 is the guilty party.
Single-locus probes will bind to just one complementary sequence
in the haploid genome. Thus, two bands will be visible in the resulting
autoradiograph; one from the paternal chromosome and one from the
maternal chromosome. This gives a simple pro?le that is often suf?-
cient to demonstrate an unambiguous match between the suspect and
the reference. The result of a paternity test using a single-locus probe
is shown in Fig. 12.10. Sometimes two or more probes can be used to
increase the number of bands in the pro?le. Single-locus probes are
more sensitive than multi-locus probes and can detect much smaller
amounts of DNA. Usually both single-locus and multi-locus probes are
used in any given case, and the results combined.252 GENETIC ENGINEERING IN ACTION
matching
band patterns
V 1 2 3
suspects suspects
4 6 7 5
Fig. 12.9 A DNA pro?le prepared using a multi-locus probe. Samples of the suspect’s
DNA isolated from the victim (V; boxed) and seven candidate suspects (1–7) were cut
with a restriction enzyme and separated on an agarose gel. The fragments were blotted
onto a ?lter and challenged with a radioactive probe. The probe hybridises to the target
sequences, producing a pro?le pattern when exposed to X-ray ?lm. The band patterns
from the victim’s sample and suspect 5 match. Courtesy of Cellmark Diagnostics.
Reproduced with permission.
12.4.3 Mysteries of the past revealed by genetic detectives
Another interesting development and application of rDNA technology
has been in examining the past -- a sort of ‘genetic history’ trail.
Although the book (and subsequent ?lm) Jurassic Park perhaps was a
little too fanciful, it is surprising how some of the ideas portrayed in
?ction have been used in the real world.
Identifying individuals using DNA pro?ling can go much further
back in time than the immediate past, where perhaps a recently
deceased individual is identi?ed by DNA analysis. In the 1990s, DNA
analysis enabled the identi?cation of notable people such as Joseph
Mengele (of Auschwitz notoriety) and Czar Nicholas II of Russia,
thus solving long-running debates about their deaths. DNA has also
been extracted from an Egyptian mummy (2400 years old) and a
human bone that is 5500 years old! Thus, it is possible to use rDNAMEDICAL AND FORENSIC APPLICATIONS OF GENE MANIPULATION 253
children
paternal
bands
maternal
bands
DF
M 1 2 3 4 F
Fig. 12.10 A DNA pro?le prepared using a single-locus probe for paternity testing.
Samples of DNA from the mother (M), four children (1–4), and the father (F) were
prepared as in Fig. 12.9. A single-locus probe was used in this analysis. The band patterns
therefore show two maternal bands and two paternal bands, one band from each
homologous chromosome on which the target sequence is located. In the case of child
1, the paternal band is different from either of the two bands in lane F, indicating a
different father (band labelled DF). This child was in fact born to the mother during a
previous marriage. Courtesy of Cellmark Diagnostics. Reproduced with permission.
technology to study ancient DNA recovered from museum specimens
or newly discovered archaeological material. Topics such as the migra-
tion of ancient populations, the degree of relatedness between differ-
ent groups of animals, and the evolution of species can be addressed
if there is access to DNA samples that are not too degraded. This
area of work is sometimes called molecular paleontology. With DNA
having been extracted from fossils as old as 65 million years, the
‘genetics of the past’ looks to provide evolutionary biologists, tax-
onomists, and paleontologists with much useful information in the
future.
Use of human remains in the identi?cation of disease-causing
organisms can also be a fruitful area of research. For example, there
In addition to medical and
forensic applications, the use of
DNA pro?ling and identi?cation
techniques is proving to be of
great value in many other areas
of science.
was some debate as to the source of tuberculosis in the Americas -- did
it exist before the early explorers reached the New World, or was it a
‘gift’ from them? By analysing DNA from the lung tissue of a Peruvian
mummy, researchers found DNA that corresponded to the tubercule
bacillus Mycobacterium tuberculosis, thus proving that the disease was
in fact endemic in the Americas prior to the arrival of the European
settlers.
In addition to its use in forensic and legal procedures, and in trac-
ing genetic history, DNA pro?ling is also a very powerful research tool
that can be applied in many different contexts. Techniques such as
RAPD analysis and genetic pro?ling are being used with many other
organisms, such as cats, dogs, birds, and plants. Application of the254 GENETIC ENGINEERING IN ACTION
technique in an ecological context enables problems that were pre-
viously studied by classical ecological methods to be investigated at
the molecular level. This use of molecular ecology is likely to have
a major impact on the study of organisms in their natural environ-
ments and (like molecular paleontology) is a good example of the
coming together of branches of science that were traditionally treated
as separate disciplines.


المادة المعروضة اعلاه هي مدخل الى المحاضرة المرفوعة بواسطة استاذ(ة) المادة . وقد تبدو لك غير متكاملة . حيث يضع استاذ المادة في بعض الاحيان فقط الجزء الاول من المحاضرة من اجل الاطلاع على ما ستقوم بتحميله لاحقا . في نظام التعليم الالكتروني نوفر هذه الخدمة لكي نبقيك على اطلاع حول محتوى الملف الذي ستقوم بتحميله .
download lecture file topic