BIOLOGY

DNA FINGERPRINTING
INVESTIGATORY PROJECT

Abstract
DNA fingerprinting is a potent new forensic technology that many consider to be the most

important tool in forensic science's history. However, as is often the case with new

technologies, societal acceptance was not easy. This project analyses this technology,

describing how it works, its applications, and its indirect journey to courtroom acceptance.

DNA Fingerprinting Types

One of the most powerful identification technologies we have for recognising an individual
or live entity is DNA fingerprinting. Except for identical twins, triplets, and other genetically
similar creatures, every living thing is genetically unique in its own way. For living
organisms, DNA is analogous to a serial number.

Each individual has a distinct sequence that is unique to that particular organism. Unlike
traditional fingerprints, which may be surgically altered or self-mutilated, the DNA
sequence can't be modified once it's left at a crime scene, making it more useful in
forensics and boosting the chances of discovering an exact match.

This form of identification can be used in a variety of applications, including forensics,

paternity testing, and molecular archaeology, all of which will be discussed later in this
chapter. To better comprehend DNA fingerprinting, we must first go through the
fundamentals of DNA.

Introduction to DNA Basics

DNA, also known as deoxyribonucleic acid, is made up of a unique sequence of bases called

nucleotides that hold all of the information about a live organism's properties. This

information was passed down to them through their parents' DNA. DNA can be found in

practically every living organism's cell. The DNA serves as a "instruction manual" for

creating living beings. Adenine (A), which bonds exclusively with thymine (T), and guanine

(G), which bonds exclusively with cytosine (C), are the four nucleotides that make up DNA

sequences. The molecular structure of DNA can be thought of as a zipper, with each tooth

representing one of the four letters (A, C, G, or T) and opposite teeth forming one of two

pairs (AT or GC).

During the division phase of a cell, a chromosome is the observable state of genetic

material. Humans contain 46 unique chromosomes, which are divided into 23 pairs. The

mother provides half of an individual's chromosomes, while the father provides the other

half. Chromosomes are linear strands of DNA that are present in the nucleus.The DNA

molecule is twisted around itself, and the super-coiled molecule is surrounded by proteins

that help it keep its shape. The genes that make each person unique are carried on the

chromosomes.

RFLPs and VNTRs

2
RFLPs, VNTRs, and STRs are the three forms of DNA fingerprints. Restriction fragment

length polymorphisms, or RFLPs, were the first type of DNA fingerprinting to appear in the

mid-eighties. The size disparities of particular genomic loci are the focus of RFLPs.

Obtaining and isolating DNA is the initial stage in producing an RFLP fingerprint. DNA may

be extracted from nearly all of the human body's cells and tissues. To supply enough DNA

for analysis, you don't need a lot of tissue or blood. The DNA is then extracted from the

blood or tissue sample, and we proceed to the cutting, size, and sorting of the DNA sample,

which is the second phase in the procedure. Restriction enzymes, which cut the DNA at

specified points, are used to cut it. Restriction enzymes are typically obtained from bacteria

that employ them to break down foreign DNA, such as viral DNA. Restriction enzymes each

identify and cut a certain DNA sequence.

At this point, the DNA is split into a variety of pieces, which are then sorted by size using a

technique known as electrophoresis. The DNA particles are combined with a buffer

solution and put to a seaweed agarose gel in this procedure. An electrical current is

connected to each side of the gel. Because the phosphate groups in DNA are negatively

charged, it migrates to the positive electrode, or anode. The fragment separation is based

on the fact that smaller bits of DNA travel faster (sieve) through the gel than bigger ones.

“This technique is the DNA equivalent of screening sand through progressively finer mesh

screens to determine particle sizes” (Betsch, 2005).

The DNA generates a band pattern in the agarose gel, which is then transferred to a nylon

sheet. A nylon sheet is laid on the gel and permitted to soak overnight in a high salt

solution to complete the transfer. The nylon membrane contains the same pattern of DNA

as the original gel when the soaking step is done. The membrane is now ready to go

through the probing process. The nylon membrane is hybridised with radioactive or

fluorescently tagged probes that attach to specific DNA sequences in the pattern, resulting

in a pattern of bands that creates the DNA fingerprint. This technique can be carried out

3
simultaneously with multiple distinct probes to produce a final product that looks quite

similar to the bar codes found in retail establishments.

VNTRs, or variable number tandem repeats, are particular places on a chromosome where

9-80 or more base tandem repeats repeat a varying number of times amongst individuals.

Using the RFLP method and a probe specific to the VNTR locus, these DNA regions may be

easily examined. The fragments are a little shorter (approximately 1-2 kilo base pairs) than

RFLPs, but they are made in the same way.

Since RFLPs and VNTRs are created in the same fashion, they exhibit the same overall

advantages and disadvantages. Some of the benefits of these DNA fingerprints include the

fact that they are the most stable and reproducible, which is a valuable quality to have

when trying to identify an exact match of a person's DNA, which must eliminate billions of

other people's DNA with a high degree of certainty. Because the DNA sample is larger than

with other forms of DNA fingerprints, contamination is easier to avoid, and little levels of

DNA contamination have no effect on the result. RFLPs and VNTRs have a number of

drawbacks, including being time consuming (especially the probe hybridization step),

requiring huge amounts of DNA to obtain an appropriate sample, and perhaps having too

many polymorphisms for a brief probe.

STRs and PCR

Short tandem repeats, or STRs for short, are currently the most used technique of DNA

fingerprinting. STRs use microsatellites with repeat sequences of only 2-5 base pairs, as

opposed to VNTRs, which study minisatellites with repeat sequences of 9-80 base pairs,

bringing the “less is more” mentality to the field of DNA fingerprinting. Because the length

of the DNA fragment being tested is short enough to be amplified by polymerase chain

reaction (PCR), we can now evaluate a very little amount of DNA in a way that is faster and

easier than any previously known approach and match it to a person's identification.

4
PCR was invented in the mid-eighties and works by using the same principles that cells use

to copy DNA to amplify a specific region, which is usually between 150 and 3,000 base pairs

long. To amplify the DNA sequence, a pair of short priming sequences (which are

complementary to the ends of the targeted sequence), a heat-resistant DNA polymerase

called Taq polymerase, a special heat-resistant DNA polymerase called Taq polymerase are

used, 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.

After that, the DNA is amplified (or reproduced) by repeating a cycle that includes three

crucial steps:

• The solution is heated to 95°C to unzip the double helix DNA structure

• The solution is cooled to 55°C to allow the primers to bind to the DNA ends

• The solution is then reheated to 75°C, which is the ideal temperature for the Taq

polymerase to make new copies of each DNA strand.

It takes about 2 minutes to complete a PCR cycle. Each cycle doubles the number of

targeted sequences in the test tube, thus hundreds of thousands of DNA copies can be

produced in as few as 50 cycles. The band amplified will represent the STR locus if primers

are chosen to flank a STR site, and the band length will be determined by a simple gel or

column.

As a result, unlike the RFLP/VNTR techniques, this procedure does not require the lengthy

probe hybridization step to the membrane. Because the entire PCR process takes only a

few hours, compared to RFLP/VNTR probe hybridization and film exposure, which can take

several days, STRs are currently the most prevalent type of DNA fingerprint. STRs can build

fingerprints from far smaller quantities of DNA than RFLPs/VNTRs, and they can even use

partially degraded DNA. As a result, the integrity and quality of the DNA sample are less

important with STRs than with other DNA fingerprinting approaches. The current standard

5
forensic technique examines 13 core STR loci that were hand-picked for their

distinctiveness.

The only downside of the STR strategy is that it is sensitive to contaminated DNA; therefore,

if contamination is suspected and enough DNA is available, the STR approach is usually

utilised first, followed by a VNTR analysis.

Applications of DNA Fingerprinting

All across the world, DNA fingerprinting is employed in a range of applications. They can be

used to solve criminal cases like rape, perform paternity tests, and even check the

authenticity of rare sports memorabilia. Whatever the case may be, DNA fingerprinting has

unquestionably changed the way the world recognises biological matches. We will discuss a

few examples of these applications and their importance below

The investigation of sexual assault and rape cases was one of the first acknowledged uses

of DNA fingerprinting. To discover who committed the crime, detectives only had to match

the DNA of the semen collected at the crime scene with the DNA of any prospective

suspect. A simple vaginal swab from the victim or any other semen discharged in the region

during the assault could yield a DNA sample from the rapist.

The figure below shows how a DNA fingerprint can help determine who is guilty of a sexual

assault.

(get better quality)

6
As can be seen in the figure, suspect B (lane 4) is guilty of rape because his DNA matches

the semen found on the victim's garments (lane 3) and in the vagina (lane 6). Because his

DNA pieces do not match the semen detected on the victim's garments or the semen from

the vaginal swab, Suspect A (lane 2) is clearly not the rapist. In such cases, DNA

fingerprinting is particularly beneficial since it gives the police a perfect match of who left

evidence at the crime site.

Paternity testing is another DNA fingerprinting application that has become widely used

around the world. In paternity tests, prospective dads of the child's DNA is compared to the

child's and mother's DNA to determine which of the potential fathers shares the most DNA

with the child. The figure shows an example of a RFLP used to determine which potential

father (F1 and F2) is the real father of the child (C). The second father tested (F2) seems to

have more DNA in common with the child than that of the first father tested (F1).

Another application of DNA fingerprinting is in molecular archaeology, which is a relatively

modern technology. DNA is used in archaeology to determine the species of an

archaeological find or to trace the bloodlines of animal or human remains. DNA can be

retrieved from a variety of sources, including biological remnants, hair, teeth, body tissues,

and even fossils. Extremely cold temperatures and arid regions are ideal for preserving

DNA. The “Tyrolean Ice-Man,” discovered in the Alps, and Egyptian mummies discovered in

the dry desert are two examples of specimens from these regions. The iceman was

discovered to be roughly 5300 years old, and DNA was retrieved from his intestinal

remnants, which revealed microscopic traces of food he ate. This was one of the most

significant archaeological finds of the twentieth century.

7
Even in the field of sports collectibles, DNA fingerprinting is used. With sports collectors

paying exorbitant prices to own a piece of sports history, there needed to be a mechanism

to verify the authenticity of the valuable items. The memorabilia can be treated with a

synthetic DNA smear, which coats the object with a hidden DNA sequence before

destroying the original batch of DNA. The collectible can then be auctioned off, providing

buyers with assurance that the item is genuine. This is only one example of how DNA

fingerprinting is employed in the modern world.

DNA Forensics

The technique of piecing together a crime scene in order to identify how the crime was

perpetrated and who was guilty is known as forensic science. DNA evidence is one of the

most well-known types of evidence employed in today's judicial system. Simply because

techniques exist to test DNA at a crime scene does not ensure that evidence was collected

correctly to avoid contamination or preserved correctly to avoid DNA decay. Many times,

inappropriate treatment of DNA evidence has prevented it from being used in a particular

prosecution.

DNA evidence may be extracted from practically any biological sample left at the crime

scene using a variety of methods. When someone committed a crime such as sexual

assault in the past, there was no meaningful way of proving their guilt unless there were

witnesses. Normal blood types are not that exclusive. With DNA forensics, a level of

confidence that is accepted as valid evidence in a criminal case may now be obtained,

whether for the prosecution or the defence. There have been countless cases in the past

where men were charged with rape and had DNA from the crime site examined, only to

discover that they were completely innocent.

8
Ways to Prevent Contamination

Contamination is one of the most serious threats to the integrity of the evidence. If your

sample of evidence is discovered to be contaminated, it can be tossed out of court.

Contamination can happen at the crime scene, during packaging, during transport to the

lab, and even during analysis. Because there is a potential of contamination in all of these

steps of the forensic process, sufficient precautions must be taken to avoid contaminating

the DNA sample.

Many things must be examined at the crime scene in order to avoid contamination. Mother

Nature is the first factor to consider. The elements can play a significant influence in

destroying evidence at a crime scene. If it rained at the murder site, for example, a blood

stain observed could be diluted, making analysis nearly difficult. Furthermore, if it had been

windy that day, critical DNA fragments could have been blown away from the crime site.

Another consideration at the crime scene is appropriately protecting the area so that the

evidence is not tainted. Many people not connected to the crime scene may have left DNA

around critical evidence that could be misinterpreted for a prospective suspect until the

crime scene is secured. Another aspect that must be regulated to prevent the possibility of

evidence contamination is equipment. Clothing, notepads, photography equipment, and

crime scene kits must all be properly decontaminated before leaving a crime scene;

otherwise, they may taint evidence at a subsequent crime scene. A mask, jumpsuit, gloves,

booties, and head cover should all be worn as disposable personal protective equipment

(PPE). Contamination at a crime scene should be kept to a minimum if these guidelines are

followed.

Conclusions

9
The most advanced method of identifying living organisms is DNA fingerprinting. DNA is a

one-of-a-kind bit of genetic material found in living organisms with one-of-a-kind features.

When DNA is left at a crime scene or deposited with a mummy, it cannot be easily

manipulated, making it a powerful forensic tool.

Traditional methods of fingerprinting DNA, such as RFLPs and VNTRs, need a reasonably

large sample and use probe hybridization to discover variations in the DNA. The most

recent form of DNA fingerprinting is STRs, which is based on PCR and takes a very minimal

amount of DNA.

DNA fingerprinting can be used in a variety of situations, including criminal rape cases,

paternity tests, molecular archaeology, and sports memorabilia. The DNA molecule is

similar to a snowflake in that no two are precisely the same, but it is one of the few things

that all living beings have in common.

One of the most useful tools in piecing together a crime scene is DNA forensics. Many

breakthroughs in the ways of collecting and maintaining these DNA samples have been

made in the last 10 years, which has aided in the acceptability of this evidence in the

courtroom. Forensic science has taken a giant stride forward in permitting DNA samples to

be used as credible evidence in courts by preventing contamination and properly keeping

them to prevent degradation. One of the most powerful weapons in determining who is

accountable for a crime is DNA evidence. With offenders altering their fingerprints and

other physical traits, DNA evidence is one of the only reliable ways to identify someone.

Crime scenes that initially appear to have no physical evidence are now investigated on a

particle level with the use of chemicals like luminol, making it nearly hard to leave a crime

scene without leaving a trace.

Although there are still some elements that make it difficult to retain a decent DNA sample,

advances in the field of forensic science will continue to be made, as it appears to have an

endless future in technology.

10
References

1. Andrews v. State of Florida (1988) District Court of Appeal of Florida, Fifth District, 533,

Southern Series, 2d, pp. 841.

2. Baldwin, Hayden B. "Crime Scene Contamination Issues." Criminal Justice Institute. 2005.

Fall 2005 <http://www.cji.net/CJI/CenterInfo/fscec/ Contamination.htm>.

3. 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.

http://papers.ssrn.com/paper.taf?abstract_id=262034

4. Betsch, David (2005) DNA Fingerprinting in Human Health and Society.

http://www.extension.iastate.edu/Publications/NCR550.pdf

5.Blackmun, J. (2004) Daubert v. Merrell Dow Pharmaceuticals, Inc. US, Legal Information

Institute, Cornell Law School. http://supct.law.cornell.edu:8080/supct/html/92-102.ZS.html

6.Coleman, Howard and Swenson, Eric (2003) “DNA in the Courtroom.” DNA in the

Courtroom: A Trial Watcher’s Guide.

<http://www.genelex.com/paternitytesting/paternitybook5.html>.

11