Biology Project

work
Topic: - DNA
Fingerprinting

AISSCE
SESSION 2019-20
Supervised By- Presented By-
 Mrs. Aruna Tripathi Ayush Kumar Singh
PGT Biology Class- XII B
A. F. S. Gorakhpur Roll No:-

CERTIFICATE
This is to certify that Ayush Kumar Singh

student of Class XII-B of AIR FORCE SCHOOL,

Gorakhpur has completed his project under my

guidance.

He has taken proper care and showed

interest & sincerity in the completion of this

project.

I certify that this project is up to my

expectations & as per the guidelines issued by

the CBSE.

Internal Examiner External Examiner Principal

ACKNOWLEDGEMENT

I express my deepest sense of

gratitude to Mrs. Aruna Tripathi
(PGT Biology) whose constant
support, guidance and inspiration has
helped me to complete this project.
I am grateful to Principal Mr. Anil
Kumar Tripathi for providing the
necessary laboratory facilities for
carrying out the work.

Ayush Kumar Singh

Class: XII-B
Roll No.:
School: Air Force
School, GKP
 ABSTRACT

DNA fingerprinting is a powerful

new forensic technology that many
argue is the greatest tool in the
history of forensic science. But as is
often the case for 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.
 TABLE OF CONTENTS

1 Abstract
2 Table of Content
3 Project Objective
4 DNA Fingerprinting Types and
Applications
5 DNA Forensics
6 Bibliography
 PROJECT OBJECTIVE

The purpose of this IQP was to

document the impact of a new
technology on society. The topic
chosen was DNA fingerprinting; that
many argue is the greatest tool in the
history of forensic science. It was
chosen in part because of its powerful
technology, well worth investigating,
and in part because of its ongoing
controversy in the courtroom, thus its
impact on society could be
documented.

DNA FINGERPRINTING TYPES

AND APPLICATIONS
DNA fingerprinting is one of the greatest
identification systems we have to recognize an
individual or 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 on in this chapter. To further understand
DNA fingerprinting we must first discuss the
basics of DNA.

Introduction to DNA Basics

DNA, also known as deoxyribonucleic acid,
contains 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 6
guanine (G) which bonds exclusively with
cytosine (C). The molecular structure of DNA
can be imagined as a zipper (Figure-1) 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
(Betsch, 2005).
Figure 1. Schematic diagram of DNA structure. Part “a”
shows a zipper which symbolizes the DNA structure. Part “b”
shows a sequence of DNA in which one side bonds with the
opposite side (Betsch, 2005).

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.
 RFLPs and VNTRs

Now that we have a better understanding of

what DNA actually is, lets 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-1980. RFLP’s 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.
The DNA at this point is cut into a various array
of pieces which are sorted according by 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 a
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 which
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. Figure 2 shows an actual RFLP-
type DNA fingerprint.

Figure 2. An example of a RFLP autoradiograph (RFLP

Autorad Image, 2003).

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 kilo base
pairs), but are created through the exact same
process. Figure 3 shows an example of a VNTR
fingerprint.

Figure 3. An example of a VNTR autoradiograph

(Texas Tech, 2005).

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 does not alter the analysis.
Some of the disadvantages of RFLPs and
VNTRs include 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 (RFLPs, 2004).
 STRs and PCR
Currently, the most popular method of DNA
fingerprinting are 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 DNA fragment being analyzed is short
enough to be amplified by polymerase chain
reaction (PCR), so now we are able to 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 1980’s and used the
same principles that cells use to replicate DNA
to amplify the specified region, which is usually
between 150-3,000 base pairs in length.
In order to amplify the DNA sequence, a pair of
short priming sequences (which are
complimentary 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
together 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. 4A).
 The solution is cooled to 55°C to allow the
primers to bind to the ends of the DNA (Fig-
4B).
 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 (Fig-5C).

Figure 4. The three steps of the PCR cycle

(Genetic Analysis, 2004).

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 (Genetic Analysis, 2004). 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. Thus this procedure avoids the
lengthy probe hybridization step to 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 than 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 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.
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, used
to conduct a paternity test, or even used 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.
 Figure 5. DNA fingerprinting used to solve a rape case (Miami, 2004).

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 committed 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. The figure-6 below shows how a DNA
fingerprint can help determine who is guilty of
a sexual assault.
As seen from figure-5, 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
crime scene.
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 and mother’s DNA in order to see
which of the potential fathers has the most
DNA in common with the child in question.
Figure 6 shows an example of a RFLP used to
determine which potential father (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 that of the first father tested
(F1).

Figure 6. A paternity test using the RFLP technique (Paternity Test, 2005).

Another application of DNA fingerprinting is a

more recent method in molecular archeology.
This method of archeology uses DNA to
determine a species of an archeological
discovery or to trace blood lines 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 dry desert. The ice man was
found to be around 5300 years old, and DNA
was extracted from the remains of his gut
which found small traces of food that he ate
(Ice Man, 2005). This was 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 7 shows
a picture of the “Tyrolean Ice-Man”.

Figure 7. The skeleton of a Neolithic man found in the Alps in 1991 (Ice Man, 2005).
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.

DNA FORENSICS
Forensic science is the art of piecing together a
crime scene in order 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.
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 8 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.

Figure 8. In the example shown on the left, DNA collected at the scene of a crime is
compared with DNA samples collected from 4 possible suspects. The DNA has been cut into
smaller pieces which are separated on a gel. The fragments from suspect 3 match those left at
the scene of the crime, betraying the guilty party (Smith, 2004).
Forensic DNA and Collection Methods
DNA can be obtained from traces of biological
fluids or tissues at a crime scene. The most
traditional sources of forensic DNA come from
saliva, seminal fluid, blood, and hair. DNA from
saliva can be extracted from items such as
cigarette butts, ski masks, envelopes, and
stamps. Seminal fluids are usually found from
oral, rectal, or vaginal swabs, and also on
clothing. Blood stains and hair that are visible
at a crime scene usually contain DNA that can
be analyzed at the forensic laboratory (Kramer,
2002). Collecting the DNA during a forensic
investigation may be the most crucial step in
terms of the analysis results. The first step in
collecting evidence is searching for it. When
examining a crime scene for evidence, a plan
should be devised to insure complete coverage
of the area. For indoor crime scenes, the area
will be searched room-by-room, with each
room divided into sectors. This examination
technique is called the zone method. For
outdoor areas, searches are conducted by
covering the area in parallel rows, until the
whole crime scene has been investigated. This
technique is called the grid method.
When collecting DNA from a crime scene it is
ideal to obtain the original item that contains
the evidence. For example, if you were
investigating a sexual assault it would be ideal
to collect undergarments worn after the
incident rather than swabbing the original
evidence (Kramer, 2002). This is not always
possible to do when the original item
containing the DNA evidence is too large to
collect, such as a sofa, but patches of covering
can be collected. For floors, individual tiles can
be collected. In order to successfully swab the
DNA you must use a cotton tipped swab and
slightly moisten the tip with clean water. Then
once the stain is absorbed by the swab let it air
dry. Do not dry the sample by any other means
because the heat could denature or
contaminate the DNA sample. After the sample
is dried package the cotton swab separately in
paper and keep the sample out of direct
sunlight (Kramer, 2002). Another method used
for collection of DNA material at a crime scene
is a tape lift. This is primarily used for dried
blood samples which are fixed to non-porous
surfaces. Traditional fingerprint tape can be
used to do this, which is then placed sticky side
down onto a piece of white paper. The tape lift
should then be sealed in a separate envelope
to prevent any contamination. When collecting
DNA evidence at a crime scene investigation,
the forensic scientist should also collect control
samples to assure that the sample is pure.
Control samples are specimens of any foreign
substance or material that may have
contaminated the DNA. The control samples
can then be analyzed separately from the other
evidence to determine if any contamination
exists.
When examining a crime scene for DNA one
must remember to wear protective gloves and
any other necessary protection in order to
prevent contamination of the sample, and also
to protect you from any possible diseases.
Other kinds of collection methods for evidence
include obtaining the original items at the
scene that the actual sample is on.

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). Another
factor at the crime scene is properly securing
the area so that people do not taint 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.
During packaging and transport of the DNA
evidence, there are also a few factors to keep
in mind in order to preserve the sample in its
original state. When packaging evidence, each
sample should be sealed individually to prevent
cross-contamination. Biological fluids should be
dried in order to avoid contamination by
bacteria (Baldwin, 2005). Evidence should be
packaged in a paper container, which is then
put into an open plastic container to reduce the
risk of evidence leaking out while in transit.
When the biological sample is in transit certain
precautions must be taken. DNA is sensitive to
temperature which means that while the
sample is in transit, the package must be out
of direct sunlight or any other extreme
circumstance. Proper arrangements must be
coordinated before transportation so that the
evidence can be properly stored until it is
ready to be analyzed.
During analysis of the DNA evidence in the
laboratory, a few things must be considered
that will aid in achieving optimal analysis.
When the sample of evidence is in temporary
storage, it should be examined to determine if
any other samples around it might have leaked
on to the sample. Proper procedures should be
followed as stated by the laboratory’s
guidelines.
Storage Methods of DNA

Biological materials are very sensitive to the

conditions around them, so storage of DNA is
an important part of maintaining useful
evidence. The samples acquired from the crime
scene should be kept away from high
temperatures. Room temperature is
acceptable, refrigeration is desirable, and
freezing is preferred when it comes to storing a
sample. Figure 9 shows a DNA freezer that is
used to store samples.

Figure 9. DNA freezer used to store

samples Gorgeous Genome, 2004).

If the samples are not stored to proper

specifications the DNA will deteriorate and will
not be able to provide proper analysis.
Freezers that are “frost-free” are the most
ideal when it comes to storage equipment.

DNA at an Aged Crime Scene

At an aged crime scene, forensic scientists

attempt to retrieve DNA evidence that is the
least contaminated and decomposed. For
example, if a deceased individual is at an aged
crime scene and is showing signs of
decomposition, then blood work would not be a
practical route of obtaining a valid DNA sample
(Kramer, 2002). If hair is present on the
deceased body, the sample must be pulled
from the tissue because the tissue attached to
the hair root is necessary for DNA analysis.
Bones and teeth would also serve as a suitable
sample of DNA evidence, since they take very
long to degrade. Another method to collect
useful evidence at an aged crime scene would
be to look for property that was associated
with the individual’s body such as a hairbrush
or toothbrush. Figure 10 shows a body at an
aged crime scene showing some obvious
decomposition.

Figure 10. A deceased individual at an

aged crime scene showing obvious stages
of decomposition (Dead Body, 1997).
Advances in Forensics
Over the past ten years there have been many
advances in DNA collection techniques that
allow this type of evidence in the courtroom
today. Standards have been set on all aspects
of crime scene investigations that help create
consistency with all evidence. DNA is now
allowed in the courtroom due to the techniques
discussed in the chapter to properly collect,
transport, analyze, and store the DNA sample
without contamination or degradation. When a
sample of DNA is kept in its original state it
proves to be one of the most reliable pieces of
evidence in the courtroom. Now we will discuss
technological advancements in the world of
forensics.
When examining a crime scene it may seem
that the lawbreaker got away without a trace.
In the past, violent crimes have been
committed and cleaned up leaving no visible
proof of the crime. We now know that tiny
particles of biological fluids, most notably
blood, can cling to most surfaces at the crime
scene for many years without anyone ever
realizing they are there (Harris, 2005). There is
now a chemical that allows you to see these
microscopic particles of blood and find the
clues, that to the naked eye, did not seem to
exist. The chemical is called luminal and it
reveals these particles with a light-producing
chemical reaction between hemoglobin and
several chemicals (Harris,
2005). Light is produced because the reactants
contain more energy than the products of the
reaction, which release the extra energy in the
form of light photons. This is the same process
that makes fireflies glow, which is called
chemiluminescence. After the chemical is
sprayed onto the object in question, a blue
glow will emit in any areas where there are tiny
traces of blood, an example of which is shown
in figure 11.

Figure 11. A simulation of luminol at

work. The chemical binds to the blood
and emits a blue glow (Harris, 2005).

This technique is used typically at violent crime

scenes, where the suspect could have
murdered the victim and got rid of all visible
evidence from the area. Once the luminol
reacts with the small traces of blood left
behind, forensic investigators will takes
pictures or videotape the crime scene to record
any potential patterns (Harris, 2005). It should
be noted that once blood reacts with luminol,
that sample is no longer able to provide DNA
for analysis. Perhaps in the future a new
method of visualization will be devised that
maintains DNA structure.
 Bibliography

In completing the project I took help of my teacher Mrs. Aruna Tripathi

& the following references

[Link]
[Link]
[Link]
APC Biology Lab
Manual for Class-XII