INDEX
Introduction 6
DNA Fingerprinting Types 7
Introduction to DNA Basics 8
RFLPs & VNTRs 10
STRs & PCR 14
Applications of DNA
17
Fingerprinting
DNA Forensics 21
Ways to Prevent
22
Contamination
Conclusions 24
Bibliography 26
5
INTRODUCTION
DNA fingerprinting is a powerful new forensic technology
that has created a revolutionary impact in crime investigation.
It is the greatest tool in the history of forensic science. DNA
fingerprinting is a laboratory technique used to determine the
probable identity of a person based on the nucleotide
sequences of certain regions of human DNA that are unique
to individuals. DNA fingerprinting is used in a variety of
situations, such as criminal investigations, other forensic
purposes, and paternity testing. In these situations, one aims
to “match” two DNA fingerprints with one another, such as a
DNA sample from a known person and one from an unknown
person. I think a lot of people are first introduced to DNA
fingerprinting while watching crime shows. An officer collects
some samples from the crime scene. They put it in a tube.
And then an hour later, they hold up a brightly colored gel,
squint at it, and say, “Aha, we have a match for the killer's
DNA”. Then the show is over. Of course, that isn't exactly
how things work in real life. But DNA fingerprinting is an
important part of forensic science. Although it can't really tell
you exactly who committed a crime, it can be used to help
narrow down a list of suspects based on how well their DNA
matches the samples that were found at the crime scene.
Investigators can also use the DNA results to search specific
databases to find other potential suspects. But like other
new technologies, its acceptance by society was not
straightforward. This project investigates this technology,
describing how it is done, its uses, and its indirect path of
acceptance in the courtroom.
6
DNA FINGERPRINTING
TYPES
DNA fingerprinting is one of the greatest identification
systems we have to recognize an individual or a living
organism. Every living creature is genetically different in its
own way, except for identical twins, triplets, etc. DNA is
comparable to a serial number for living things. Each
individual contains a unique sequence that is specific to that
one organism. Unlike traditional fingerprints, which can be
surgically altered or self-mutilated, the DNA sequence
cannot easily be changed once the material is left at a crime
scene, thus increasing its effective use in forensics and the
probability of finding an exact match. This method of
identification is useful in many applications, such as
forensics, paternity testing, and molecular archeology, which
we will discuss later. To further understand DNA
fingerprinting, we must first discuss the basics of DNA.
Figure: 1. Fingerprints.
7
Introduction to DNA
Basics
DNA, also known as deoxyribonucleic acidcontains a
specific sequence of bases called nucleotides, which
contain the information of all the characteristics of living
organisms. This information was inherited through the DNA of
their parents. DNA is found in almost every cell of every living
organism. The DNA represents the “instruction book” for
making living organisms. The four nucleotides that constitute
the sequences of DNA are adenine (A), which bonds
exclusively with thymine (T), and guanine (G), which bonds
exclusively with cytosine (C). The molecular structure of DNA
can be imagined as a zipper Figure 22) with each tooth
representing one of the four letters (A, C, G, or T) and with
opposite teeth forming either of the two pairs, AT or GC.
Figure: 2. Molecular structure of DNA.
8
A chromosome is the visible state of genetic material
during the division phase of a cell. Humans have 23 pairs of
chromosomes, which makes 46 individual chromosomes.
Half of the chromosomes of an individual come from the
mother and the other half from the father. Chromosomes
are found in the nucleus and contain a linear strand of DNA.
The DNA molecule is twisted onto itself, and the super-
coiled molecule is enclosed in proteins, which help
maintain its shape. The chromosomes carry the genes that
make each individual.
Figure: 3. DNA & Chromosome.
9
RFLP AND VNTR
Now that we have a better understanding of what DNA
actually is, let's move on to the basics of making a DNA
fingerprint. There are three types of DNA fingerprints: RFLPs,
VNTRs, and STRs. Restriction fragment length
polymorphisms, or RFLPs as they are commonly known,
were the first type of DNA fingerprinting which came onto
the scene in the mid-1980s. RFLPs focus on the size
differences of certain genetic locations.
The first step in creating an RFLP fingerprint is obtaining and
isolating the DNA. DNA can be obtained from almost any of
the cells or tissues in the human body. You do not need a
large amount of tissue or blood to provide enough DNA for
analysis. The DNA is then extracted from the blood or tissue
sample, and from here we carry out our second step in the
process which is the cutting, sizing, and sorting of the DNA
sample. DNA is cut using restriction enzymes, which cut the
DNA stand at specific places. Restriction enzymes are
usually isolated from bacteria that use them to degrade
foreign DNA like viral DNA. Each type of restriction enzyme
recognizes and cuts a particular DNA sequence.
10
The DNA at this point is cut into a variety of pieces, which are
sorted according to size through a process called
electrophoresis. In this process, the DNA particles are mixed
into a buffer solution and applied to a gel made from seaweed
agarose. Each side of the gel is connected to an electrical
current. The DNA is negatively charged due to its phosphate
groups, so it migrates towards the positive electrode or anode.
The smaller pieces of DNA move faster (sieve) through the gel
than the larger ones, so this provides the basis of the fragment
separation. “This technique is the DNA equivalent of screening
sand through progressively finer mesh screens to determine
particle sizes” (Betsch, 2005).
The band pattern that the DNA creates in the agarose gel is
then transferred to a nylon sheet. To complete this transfer, a
nylon sheet is placed on the gel and left to soak overnight in a
high salt solution. After the soaking procedure is completed,
the nylon membrane contains the same pattern of DNA as
occurred in the original gel. The membrane is now prepared to
undergo its probing phase. Radioactive or fluorescently
labeled probes are hybridized onto the nylon membrane, which
bind to specific DNA sequences present in the pattern to
produce a pattern of bands that create the DNA fingerprint.
This process can be performed with several different probes
simultaneously to make the final product, which looks very
similar to the bar codes you see in retail stores.
11
Figure: 4. RFLP-type DNA fingerprint.
Variable number tandem repeats, or VNTRs, represent specific
locations on a chromosome in which tandem repeats of 9-80
or more bases repeat a different number of times between
individuals. These regions of DNA are readily analyzed using
the RFLP approach and a probe specific to a VNTR locus. The
fragments are a little shorter than RFLPs (about 1-2 kilobases),
but are created through the same process. Figure 5 shows an
example of a VNTR fingerprint.
Figure: 5. An example of VNTR autoradiograph.
12
Since RFLPs and VNTRs are created in the same fashion, they
exhibit the same overall advantages and disadvantages. Some
of the advantages of these types of DNA fingerprints are that
they are the most stable and reproducible, which is a valuable
trait to have when you are trying to determine an exact match
of a person’s DNA, which must exclude billions of other
people’s DNA with a certain degree of confidence. They are
also easier to prevent contamination since the DNA sample is
larger than with other types of DNA fingerprints, and small
amounts of DNA contamination do not alter the analysis. Some
of the disadvantages of RFLPs and VNTRs include that they are
very time-consuming (especially the probe hybridization step),
relatively large amounts of DNA must be used to obtain an
adequate sample, too many polymorphisms may be present for
a short probe, and the cost is very high due to labor and time
requirements
13
STR AND PCR
Currently, the most popular method of DNA fingerprinting is
short tandem repeats, or STRs for short. Unlike VNTRs, which
analyze minisatellites that have repeat sequences of 9-80
base pairs, STRs use microsatellites, which have repeat
sequences of only 2-5 base pairs, introducing the “less is
more” philosophy to the world of DNA fingerprinting. This was a
big step forward in forensic science since the length of the
DNA fragment being analyzed is short enough to be amplified
by polymerase chain reaction (PCR), so now we can analyze a
very small sample of DNA that is quicker and easier than any
previously known method and match it to a person’s identity.
PCR was developed in the mid-1980s, nd the same principles
that cells use to replicate DNA are used to amplify the
specified region, which is usually between 150-3,000 base
pairs in length. To amplify the DNA sequence, a pair of short
priming sequences (which are complementary to the ends of
the targeted sequence), a special heat-resistant DNA
polymerase called Taq polymerase, and a solution of the four
DNA bases are all mixed in a test tube, which contains a few
copies of the targeted DNA sequence (Genetic Analysis,
2004).
The DNA is then amplified (or replicated) by the repetition of a
cycle, which contains three vital steps:
• The solution is heated to 95°C to unzip the double helix DNA
structure (Fig. 6A).
• The solution is cooled to 55°C to allow the primers to bind to
the ends of the DNA (Fig. 6B).
14
• The solution is then reheated to 75°C, which is the optimal
temperature for the Taq polymerase to create new copies of
each DNA strand.
Figure: 6. Conventional PCR process.
One PCR cycle takes approximately 2 minutes to complete.
Each cycle doubles the amount of the previous amount of
targeted sequences in the test tube, so it only takes about 50
cycles to produce hundreds of thousands of DNA copies. So
long as primers are chosen to flank an STR site, the band
amplified will represent the STR locus, and a simple gel or
column will determine the band length.
15
Thus, this procedure avoids the lengthy probe hybridization
step of the membrane of the RFLP/VNTR approaches. STRs
are currently the most popular type of DNA fingerprint, since
the whole PCR process takes only a few hours, compared to
RFLP/VNTR probe hybridization and film exposure, which can
take several days. STRs can use much smaller samples of
DNA than RFLPs/VNTRs, and can even use partially degraded
DNA to create a fingerprint. Thus, the integrity and quality of
the DNA sample is not as great a factor with STRs as with the
traditional methods of DNA fingerprinting (Introduction to
STRs, 2005). The current standard forensic protocol analyses
13 core STR loci, which have been carefully chosen for their
uniqueness. The only disadvantage of the STR approach is
that it is sensitive to contaminating DNA, so usually the STR
approach is used first, followed by a VNTR analysis if
contamination is suspected, and enough DNA is available.
16
APPLICATIONS OF DNA
FINGERPRINTING
DNA fingerprinting is used in a variety of applications all over
the world. They can be used to solve criminal cases such as
rape, to conduct a paternity test, or even to determine the
authenticity of rare sports memorabilia. Whatever the case, it
is evident that DNA fingerprinting has revolutionized the way
the world identifies biological matches. We will discuss a few
examples of these applications and their importance below.
One of the first accepted uses of DNA fingerprinting was in the
investigation of sexual assault and rape cases. Detectives only
had to match the DNA of the semen found at the scene of the
crime with the DNA of any potential suspect to determine who
was guilty of committing the crime. A DNA sample from the
rapist could be obtained from a simple vaginal swab from the
victim or any other semen that was released in the area during
the assault. Figure 7 below shows how a DNA fingerprint can
help determine who is guilty of a sexual assault.
Figure: 7. Use of DNA typing to help identify a rapist.
17
As seen from figure-6, suspect B (lane 4) is guilty of rape
because his DNA fragments match that of the semen found on
the victim’s clothes (lane 3) and also in the vagina (lane 6).
Suspect A (lane 2) is clearly not the rapist because his DNA
fragments do not match the semen found on the victim’s
clothes or the semen from the vaginal swab. DNA
fingerprinting is very useful in such an application because it
provides the police with an exact match of who left evidence
at the crimescene.
Figure: 8. RFLP.
Paternity tests are another application of DNA fingerprinting
that has been incorporated around the world. In paternity
tests, potential fathers of the child have their DNA analyzed
with the child's and mother’s DNA to see which of the potential
fathers has the most DNA in common with the child in
question. Figure 8 shows an example of an RFLP used to
determine which potential fathers (F1 and F2) is the real father
of the child (C). As you can see in the figure below, the second
father tested (F2) seems to have more DNA in common with
the child than the first father tested (F1).
18
Another application of DNA fingerprinting is a more recent
method in molecular archeology. This method of archeology
uses DNA to determine the species of an archeological
discovery or to trace the bloodlines of animal or human
remains. DNA may be extracted from biological remains, hair,
teeth, body tissues, or even fossils. The best climates to
preserve DNA are very cold temperatures and arid climates.
Some examples of specimens from these types of climates
are the “Tyrolean Ice-Man”, who was found in the Alps, and the
mummies of Egypt found in the desert. The Iceman was found
to be around 5300 years old, and DNA was extracted from the
remains of his gut, which revealed small traces of food that he
ate (Ice Man, 2005). This was 19 one of the most historic
archeological discoveries in the last century. DNA
fingerprinting is an important tool for archeologists to piece
together information that links the past to us today. Figure 9
shows a picture of the “Tyrolean Ice-Man”.
Figure: 9. Tyrolean Ice-Man.
19
DNA fingerprinting is even used in the world of sports
collectibles. With sports collectors spending gigantic amounts
of money to own a piece of sports history, there needed to be
a way to validate the authenticity of the rare memorabilia. The
memorabilia can be treated with a synthetic DNA smear, in
which the item is coated with a secret DNA sequence, where
the original batch of DNA is then destroyed. The collectible
can then be auctioned off, giving the buyers assurance that
the product is indeed authentic. This is just another instance of
how DNA fingerprinting can be used in today’s world.
Figure: 10. Sports collectibles.
20
DNA FORENSICS
Forensic science is the art of piecing together a crime scene
to determine how the crime was committed and who was
responsible. DNA evidence is one of the most prominent
pieces of evidence that is used in the United States judicial
system today. Just because techniques exist that allow DNA to
be analyzed at a crime scene does not necessarily mean that
evidence was collected correctly to avoid contamination, or
was stored correctly to prevent DNA degradation. As we will
learn in Chapter 3, when we discuss landmark DNA court
cases, many times, DNA evidence has been prevented from
use in a particular trial due to improper handling. The purpose
of this chapter is to discuss some of the current knowledge
about proper DNA handling.
DNA evidence can be collected by various means from almost
any biological sample that was left at the scene of the crime.
In the past, when someone committed a crime such as a
sexual assault, unless there were witnesses, there was no real
way of proving that a specific person was guilty. Normal blood
types are not that exclusive. Now with DNA forensics, a level
of certainty can be established that is recognized as valid
evidence in a criminal case, either for the prosecution or the
defense. There have been numerous instances where men
were charged with rape in the past and had DNA analyzed
from the crime scene, only to find out that they were innocent
all along. Figure 1 shows an example of how DNA analysis can
help determine who is guilty of the crime in question. Note
how the crime scene sample matches suspect 3. We will now
discuss the proper techniques to conduct a forensic
investigation.
21
WAYS TO PREVENT
CONTAMINATION
Contamination is one of the greatest risks that the evidence
must be guarded from. If your sample of evidence is found to
be contaminated, it can be thrown out as evidence in the
courtroom. Contamination can occur at the crime scene,
during packaging, in transit to the laboratory, and also during
analysis. With a risk of possible contamination present in all
these steps of the forensic process, proper precautions must
be used to prevent ruining the DNA sample. At the crime
scene, many factors must be considered in trying to prevent
contamination. The first factor is Mother Nature. The outdoor
elements can play key roles in ruining evidence at the crime
scene. For example, if it rained at the crime scene, a blood
stain found could be diluted, which would be almost
impossible to analyze. Also, if it was windy that day, then vital
pieces of DNA could have been blown away from the crime
scene (Baldwin, 2005).
22
Another factor at the crime scene is properly securing the
area so that people do not tamper with the evidence. Until a
crime scene is secured many individuals not related to the
event may have left DNA around key evidence which may be
mistaken for a possible suspect. Equipment is another factor
which must be regulated to reduce the risk of evidence
contamination. Clothing, notepads, photography equipment,
and crime scene kits must be properly decontaminated once
leaving a crime scene or they may contaminate evidence at
another crime scene. Disposable personal protective
equipment (PPE) should be worn including: a mask, jumpsuit,
gloves, booties and head cover (Baldwin, 2005). By keeping
these tips in mind, contamination at a crime scene should be
at a minimum.
23
CONCLUSION
DNA fingerprinting is the most sophisticated way to identify
living organisms. DNA is a unique piece of genetic material
within biological organisms, which has characteristics that are
one of a kind. DNA cannot easily be altered once it is left at a
crime scene or deposited with a mummy, which makes it a
strong forensic tool. RFLPs and VNTRs are the traditional
methods of DNA fingerprinting, which use a relatively large
sample and the method of probe hybridization to detect
polymorphisms in the DNA. STRs are the most current form of
DNA fingerprinting, which is PCR-based and uses a very small
sample of DNA. DNA fingerprinting has many applications that
range from criminal rape cases, paternity tests, molecular
archeology, sports memorabilia, etc. The DNA molecule is like
a snowflake in that there are no two exactly alike, but it is one
of the only things in common that all biological organisms are
created with it.
DNA forensics is one of the greatest tools in piecing together a
crime scene. Over the past ten years, there have been many
advances in the methods of collecting and preserving these
DNA samples to help facilitate the acceptance of this evidence
in the courtroom. By avoiding contamination and properly
storing it to prevent degradation, forensic science has made a
monumental step in allowing DNA samples as valid evidence in
United States courtrooms. DNA evidence is now one of the
most powerful tools used in determining who is responsible for
a crime. With criminals altering their fingerprints and other
physical characteristics, DNA evidence is one of the only true
methods to correctly identify an individual
24
Now, with the help of chemicals such as luminol, crime scenes
that at first analysis seem to have no physical evidence are
further examined on the particle level, which makes it almost
impossible to leave a crime without a trace. Although there are
still some factors that make it difficult to preserve a good DNA
sample, progress will continue to be made in the field of
forensic science, which seems to have a limitless future in
technology to come.
Figure: 11. DNA Fingerprinting.
25
BIBLIOGRAPHY
Andrews v. State of Florida (1988) District Court of Appeal
of Florida, Fifth District, 533, Southern Series, 2d, pp. 841.
Baldwin, Hayden B. "Crime Scene Contamination Issues."
Criminal Justice Institute. 2005. Fall 2005.
Bernstein, David (2001) “Frye, Frye, Again: The Past,
Present, and Future of the General Acceptance Test.” Law
and Economics Research Papers Series Paper No. 01-07.
[Link]
Betsch, David (2005).DNA Fingerprinting in Human Health
and Society.
[Link]
Blackmun, J. (2004) Daubert v. Merrell Dow
Pharmaceuticals, Inc. United States, Legal Information
Institute, Cornell Law School.
[Link]
[Link]
Coleman, Howard, and Swenson, Eric (2003). “DNA in the
Courtroom.” DNA in the Courtroom: A Trial Watcher’s Guide.
26
THANK
YOU