DEFINITION OF DNA FINGERPRINTING

DNA fingerprinting is a scientific technique used to identify individuals

based on unique genetic patterns found in their DNA. It analyses specific
regions of the genome particularly sequences like short tandem repeats
(STRs) and variable number tandem repeats (VNTRs). It is also defined as
the comparison of genetic information of a person to evidence found at
the scene of offence or crime. This method is widely used in forensic
science, paternity testing, and genetic research. [1] (Brenner’s 2013)

PRINCIPLE OF DNA FINGERPRINTING

DNA fingerprinting is based on the uniqueness of certain regions in an

individual’s genome, particularly non-coding sequences that contain
highly polymorphic repetitive elements. The primary focus is on Short
Tandem Repeats (STRs) and Variable Number Tandem Repeats (VNTRs),
which show significant variation in length and sequence among
individuals.

The process begins with DNA extraction from a biological sample (such as
blood, saliva, hair, or tissue). Specific regions of interest are then
amplified using Polymerase Chain Reaction (PCR). Alternatively,
Restriction Fragment Length Polymorphism (RFLP) can be used, where
restriction enzymes cut DNA at specific sites, producing fragments of
varying lengths.

The amplified or fragmented DNA is then separated using Gel

Electrophoresis or Capillary Electrophoresis. In forensic and genetic
studies, the resulting banding pattern or electropherogram is analysed
and compared with other DNA samples. Since STRs and VNTRs exhibit
high variability, the probability of two unrelated individuals having the
same DNA profile is extremely low, the odds of two people, who are not
related by blood, having the exact same DNA fingerprint is about 1 in
594.1 trillion individuals, making DNA fingerprinting a powerful tool [1]
(Brenner’s 2013)

LITERATURE REVIEW
History and timeline of DNA Fingerprinting

1. Early Foundations (1960s–1970s): Discovery of DNA Variability

Before DNA fingerprinting was invented, scientists had already discovered

that human DNA contains variable regions that could be used for
identification. In the 1960s, researchers explored genetic polymorphisms,
especially in blood proteins, to differentiate individuals. However, these
techniques were limited in accuracy.

By the 1970s, scientists such as Ray White (1978) discovered the first
restriction fragment length polymorphisms (RFLPs)—DNA sequences that
vary between individuals. These variations laid the groundwork for the
idea that DNA itself could be a unique identifier. [2] (Jeffreys et al 1985)

2. The Breakthrough: Alec Jeffreys and the Discovery of DNA

Fingerprinting (1984–1985)

In September 1984, at the University of Leicester, British geneticist Sir

Alec Jeffreys made a ground breaking discovery. While studying DNA
sequences called minisatellites, Jeffreys realized that these regions were
highly variable among individuals (except identical twins). He noted
unique patterns in DNA bands on an X-ray film, leading to the birth of DNA
fingerprinting.

In March 1985, Jeffreys and his team published their findings and
demonstrated how DNA fingerprinting could be used for individual
identification.[2] (Jeffreys et al 1985)

FIRST REAL-WORLD APPLICATIONS (1985–1987)

DNA fingerprinting was first used in immigration cases and criminal

investigations:

 1985: The first practical use of DNA fingerprinting was in an

immigration case, where it helped confirm the parentage of a boy
whose family was applying to stay in the UK. This was the first legal
recognition of DNA as an identity verification tool.
 1986: DNA fingerprinting was first used in a criminal case—the
infamous Colin Pitchfork murder case in the UK. DNA analysis
proved that Pitchfork, and not an earlier suspect, was the true
perpetrator. This marked the beginning of DNA's role in forensic
science.

EVOLUTION OF DNA PROFILING TECHNIQUES (1987–1995)

While Jeffreys' method was revolutionary, early DNA fingerprinting relied
on RFLP analysis, which was time-consuming and required large DNA
samples. Scientists sought faster, more efficient techniques:

 1987: The first commercial DNA fingerprinting service was

established by Cellmark Diagnostics, allowing widespread forensic
and legal use.
 1988: The FBI first used DNA evidence in a criminal case in the US.
 1991: The shift from RFLP to Polymerase Chain Reaction (PCR)-
based DNA profiling made the process faster and more sensitive,
allowing analysis of degraded samples.
 1995: The UK National DNA Database (NDNAD) was established, the
first national database of DNA profiles for criminal investigations.[3]
(Mullis and Faloona 1987)

THE 21ST CENTURY: ADVANCES AND ETHICAL CONCERNS

(2000–PRESENT)

Modern DNA fingerprinting techniques have continued to evolve:

 2000s: Development of Short Tandem Repeat (STR) Analysis, which

is now the global standard for forensic DNA profiling.
 2003: Completion of the Human Genome Project, improving our
understanding of genetic variation.
 2010s: The introduction of Rapid DNA Technology allowed on-site
DNA analysis within 90 minutes.
 2020s: The rise of genetic genealogy (e.g., GEDmatch, used to catch
the Golden State Killer) has expanded the scope of DNA in forensic
science but raised concerns about privacy and ethics.

APPLICATIONS

1. Forensic Investigations

DNA fingerprinting plays a pivotal role in forensic science by linking

suspects to biological evidence found at crime scenes. This method has
been instrumental in both securing convictions and exonerating
individuals wrongfully accused. The technique's precision allows for the
analysis of minute biological samples, making it invaluable in criminal
investigations. [4] (Saad R 2005)
2. Paternity and Custody Cases

In legal disputes involving paternity, child custody, and child support, DNA
profiling provides definitive evidence regarding biological relationships. By
comparing genetic material, courts can make informed decisions,
ensuring that parental responsibilities and rights are appropriately
assigned. [4] (Saad R 2005)

3. Medical Diagnostics

Beyond legal applications, DNA fingerprinting is utilized in the medical

field to diagnose inherited disorders and human diseases. By analyzing an
individual's genetic makeup, healthcare professionals can identify
predispositions to certain conditions, allowing for early interventions and
personalized treatment plans. [4] (Saad R 2005)

4. Population Genetics Studies

Researchers employ DNA markers to study genetic variations within and

between populations. This application aids in understanding evolutionary
relationships, migration patterns, and the genetic diversity of human
populations, contributing to broader anthropological and genetic research.
[4] (Saad R 2005)

The Colin Pitchfork Case: The First Criminal Conviction Using

DNA Evidence
Background

In 1983 and 1986, two teenage girls, Lynda Mann (15) and Dawn
Ashworth (15), were raped and murdered in Narborough, Leicestershire,
England. The cases remained unsolved for years due to a lack of
conclusive evidence.

Breakthrough with DNA Profiling

In 1986, a 17-year-old named Richard Buckland was arrested after he

confessed to the murder of Dawn Ashworth but denied killing Lynda Mann.
Despite his confession, police sought scientific evidence to confirm his
involvement.

At the time, Alec Jeffreys at the University of Leicester had recently

discovered DNA fingerprinting. The police contacted Jeffreys and
requested his help.

 Jeffreys analyzed DNA samples from both crime scenes and

compared them to Buckland's DNA.
  The results shocked investigators—Buckland's DNA did not match
the semen samples from either crime, proving his innocence. He
became the first person in history to be exonerated through DNA
evidence.

Catching the Real Killer

Knowing the true murderer was still at large, the police conducted a mass
DNA screening. They collected over 5,500 DNA samples from local men,
but none matched.

In 1987, a breakthrough occurred:

 A man named Colin Pitchfork, who had earlier avoided testing by

persuading a coworker to take the test for him, was overheard
bragging about the deception.
 The police arrested Pitchfork and took a DNA sample.
 His DNA perfectly matched the semen samples from both crime
scenes, conclusively identifying him as the real killer. [5] (Jeffreys et
al 1985)

Conviction and Impact

 Pitchfork was convicted in 1988 and sentenced to life imprisonment.

This was the first case in history where DNA fingerprinting led to
both an exoneration and a conviction.[5] (Jeffreys et al 1985)

ADVANTAGES

Forensic Applications: DNA fingerprinting enables precise identification of

individuals involved in criminal activities, aiding in both convictions and
exonerations. Its unparalleled specificity has revolutionized forensic
investigations. [6] (Roewer L 2013)
Paternity and Kinship Testing: The technique provides accurate
determination of biological relationships, which is essential in resolving
paternity disputes and verifying familial connections.
Medical Research: DNA profiling assists in understanding genetic
predispositions to diseases, facilitating personalized medicine and
advancing genetic research. [7] (Cawood A H 1989)

Disadvantages:

1. Privacy Concerns: The collection and storage of genetic information

raise significant privacy issues. Unauthorized access or misuse of
DNA data can lead to ethical and legal dilemmas.
 2. Potential for Misuse: Improper handling or interpretation of DNA
evidence can result in miscarriages of justice, such as wrongful
convictions. The reliability of results heavily depends on stringent
laboratory protocols.
3. Ethical and Legal Challenges: The establishment of DNA databases
and the potential for genetic discrimination pose ethical and legal
challenges. Balancing the benefits of DNA fingerprinting with
individual rights remains a complex issue.

METHODOLOGY

1. Sample Collection and DNA Extraction

Buccal Swab Collection:

1. Use sterile buccal swabs to collect epithelial cells from the inner
cheek.
2. Allow the swabs to air-dry before processing.
3. DNA Extraction Using the GeneCatcher™ gDNA 0.3–1 mL Blood Kit:

Although designed for blood samples, this kit can be adapted for buccal
swabs.

Materials Needed:

 GeneCatcher™ Magnetic Beads

 GeneCatcher™ Lysis Buffer (L13)
 Protease and Protease Buffer
 Wash Buffer (W12)
 Elution Buffer (E5; 10 mM Tris-HCl, pH 8.5)
 100% Isopropanol
 50% (v/v) Isopropanol
 24-well Magnetic Separator
 Adjustable pipettes and aerosol barrier pipette tips
 Water bath or heat block set at 65°C
Procedure

Cell Lysis:

Add 180 µL of Digestion Solution to the sample.

Add 20 µL of Proteinase K solution.

Incubate at 56°C for 30 minutes to lyse cells and digest proteins.

RNA Removal:

Add 20 µL of RNase A Solution.

Incubate at room temperature for 10 minutes to degrade RNA.

DNA Binding:

Add 200 µL of Lysis Solution to the lysate.

Add 400 µL of 96–100% ethanol.

Transfer the mixture to the GeneJET spin column.

Centrifuge at 6,000 × g for 1 minute.

Washing:

Add 500 µL of Wash Buffer I to the column.

Centrifuge at 8,000 × g for 1 minute.

Add 500 µL of Wash Buffer II.

Centrifuge at 12,000 × g for 3 minutes to remove impurities.

Elution:

Transfer the spin column to a clean microcentrifuge tube.

Add 200 µL of Elution Buffer.

Incubate for 2 minutes at room temperature.

Centrifuge at 8,000 × g for 1 minute to elute the DNA.

3. DNA Quantification

Measure DNA concentration using a spectrophotometer or fluorometer to

ensure sufficient quantity and quality for downstream applications.

https://assets.thermofisher.com/TFS-Assets/LSG/manuals/
MAN0012663_GeneJET_Genomic_DNA_Purification_Kit_UG.pdf

4. PCR Amplification

Amplify specific STR loci using the GlobalFiler PCR Amplification Kit.

Materials:

GlobalFiler PCR Amplification Kit

Thermal cycler

PCR-grade water

Procedure:

1.Reaction Setup:

Prepare the PCR reaction mix on ice:

10 µL of GlobalFiler Master Mix

5 µL of GlobalFiler Primer Set

0.5–1.0 ng of template DNA

Add PCR-grade water to a final volume of 25 µL.

 PCR Cycling Conditions:

o Initial denaturation at 94°C for 2 minutes.
o 30 cycles of:
 Denaturation at 94°C for 30 seconds.
 Annealing at the primer-specific temperature for 30
seconds.
 Extension at 72°C for a time appropriate for the
amplicon length.
o Final extension at 72°C for 5 minutes.
o Hold at 4°C.

3. Agarose Gel Preparation and Electrophoresis

  Preparation of 1% Agarose Gel:
o Dissolve 1.0 g of UltraPure™ Agarose in 100 mL of 1X TAE
Buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0).
o Heat until fully dissolved; cool to ~55°C.
o Pour into a gel tray with a comb; allow to solidify.
o
 Electrophoresis:
o Place the gel in the electrophoresis chamber; cover with 1X
TAE Buffer.
o Mix 5 µL of PCR product with 1 µL of 6X DNA Loading Dye;
load into wells.
o Include 5 µL of Invitrogen™ 100 bp DNA Ladder as a size
reference.
o Run at 100 volts until the dye migrates appropriately.

4. Visualization and Analysis

 Stain the gel with SYBR™ Safe DNA Gel Stain according to the
manufacturer's instructions.
 Visualize under blue light using a gel documentation system.
 Compare band patterns to the DNA ladder to determine fragment
sizes and analyze individual DNA profiles.

https://www.thermofisher.com/us/en/home/industrial/forensics/human-
identification/forensic-dna-analysis/pcr-amplification-forensic-dna-
profiling.html

RESULTS OF DNA FINGERPRINTING

When DNA fingerprinting is performed, the results typically include:

1. Electropherogram Output
o The primary result of DNA fingerprinting is an
electropherogram, a graphical representation of DNA
fragment sizes obtained through capillary electrophoresis.
o Each peak in the electropherogram corresponds to an allele at
a specific STR (short tandem repeat) locus.
o Individuals have a unique combination of peaks across
multiple loci, forming a distinctive genetic profile.
2. Numerical DNA Profile
o The STR analysis results in a series of numbers representing
the number of repeat units at each STR locus.
o Example output:

Locus | Allele 1 | Allele 2

 D3S1358 | 15 | 18
vWA | 17 | 19
FGA | 22 | 23

o This profile can be compared with known DNA samples from

suspects, crime scenes, or databases.

Credit
B. Steffen/NIST

This DNA profile is based on 24 genetic markers.In this graph, the peaks
represent the number of repeats at each marker. Most markers show two
peaks,one inherited from each parent (where only one peak appears in a
marker, the individual has inherited the same number of repeats from
each parent). These 24 markers produce a series of 48 numbers that can
be used to uniquely identify an individual.

3. Match Probability Calculation

o If two profiles match, a statistical probability is calculated to
determine the likelihood of the match occurring by chance.
o For example, the probability of two unrelated individuals
sharing the same STR profile can be as low as 1 in a trillion,
depending on the number of loci analyzed.
4. Forensic Conclusions
 o A match between a crime scene sample and a suspect's DNA
strongly suggests involvement.
o In paternity testing, a child’s DNA profile is compared to
potential parents, and if the alleles do not match expected
inheritance patterns, exclusion is confirmed with 99.99%
certainty.
o In missing person cases, DNA profiles are compared against
relatives or databases to establish identity.

These results provide highly conclusive identification data, making DNA

fingerprinting a powerful tool in forensic science, medicine, and
genealogy. [8] (Butler J M 2015)

Discussion of DNA Fingerprinting Results

The results of DNA fingerprinting, particularly the unique genetic profiles

obtained through STR (Short Tandem Repeat) analysis, highlight the
precision and reliability of this technique in forensic and biomedical
applications. The electropherogram output, consisting of peaks
corresponding to specific STR loci, serves as a robust and highly
discriminatory tool for individual identification.

One of the most significant aspects of DNA fingerprinting is its extremely

low probability of error when comparing profiles. Studies have shown that
the likelihood of two unrelated individuals sharing the same DNA profile
across a standard set of STR markers is less than one in a billion. This high
specificity has made DNA fingerprinting a gold standard in forensic
investigations, allowing law enforcement to match crime scene evidence
with suspects conclusively.

Additionally, the use of STR-based DNA profiling in paternity testing has

revolutionized legal and medical cases. By comparing the inheritance
patterns of alleles between a child and potential parents, DNA
fingerprinting provides over 99.99% certainty in determining biological
relationships. This has had profound implications for resolving custody
disputes, inheritance claims, and immigration cases.

However, despite its accuracy, the technique is not without challenges.

The quality of DNA samples plays a crucial role in obtaining reliable
results. Factors such as DNA degradation, contamination, and the
presence of mixed samples can complicate interpretation. Moreover,
ethical concerns regarding privacy and the storage of genetic information
in national databases remain a topic of debate.

Future advancements in next-generation sequencing (NGS) and

miniaturized DNA analysis technologies may further enhance the accuracy
and efficiency of DNA fingerprinting. Emerging methods, such as Rapid
DNA technology, could allow for faster on-site profiling, making forensic
investigations more efficient and accessible. [9] (Mattei et al 2013)

CONCLUSION

DNA fingerprinting has emerged as one of the most powerful tools in

modern molecular biology, revolutionizing forensic science, paternity
testing, evolutionary biology, and biodiversity conservation. By leveraging
the variability in short tandem repeats (STRs) and other polymorphic
regions of the genome, the technique provides a precise, reproducible,
and highly individualized genetic signature. As reported in Nature, the
early development of DNA fingerprinting laid the foundation for its role in
criminal justice systems, allowing for the accurate identification of
individuals from minute biological traces and playing a pivotal role in both
convicting offenders and exonerating the innocent .

Contemporary applications, as highlighted by ScienceDirect, extend well

beyond forensics—into fields such as species classification, wildlife
forensics, and monitoring of genetic diversity. In conservation genetics,
for example, DNA fingerprinting enables the identification of poached
wildlife products and the tracking of endangered species’ populations,
thereby contributing to global biodiversity protection efforts.

Looking ahead, the integration of DNA fingerprinting with high-throughput

sequencing technologies and AI-driven analysis tools is poised to enhance
both the sensitivity and speed of genetic profiling. Future advancements
may allow for real-time DNA identification at crime scenes using portable
devices, and improved automation could increase the capacity for large-
scale database searches within seconds. These prospects not only
promise greater efficiency and accuracy but also introduce new ethical
and regulatory challenges concerning privacy, data security, and consent.

In sum, DNA fingerprinting stands as a cornerstone of genetic

identification. Its evolution continues to reshape how science intersects
with law, conservation, and society, affirming its enduring value and
expanding impact.

REFERENCES
[1] Brenner's Encyclopedia of Genetics (Second Edition), 2013
https://www.sciencedirect.com/science/article/pii/B978012374984000419
8
[2] Jeffreys, A. J., Wilson, V., & Thein, S. L. (1985). Hypervariable
"minisatellite" regions in human DNA. Nature, 314(6006), 67–73.
https://doi.org/10.1038/314067a0

[3] Mullis, K., & Faloona, F. (1987). Specific enzymatic amplification of

DNA in vitro: The polymerase chain reaction. Cold Spring Harbor
Symposia on Quantitative Biology, 51, 263–273.
https://doi.org/10.1101/SQB.1986.051.01.032

[4] Saad R. (2005). Discovery, development, and current applications of

DNA identity testing. Proceedings (Baylor University. Medical
Center), 18(2), 130–133.
https://doi.org/10.1080/08998280.2005.11928051

[5] Jeffreys, A. J., Brookfield, J. F., & Semeonoff, R. (1985). Positive

identification of an immigration test-case using human DNA fingerprints.
Nature, 317(6040), 818–819. https://doi.org/10.1038/317818a0

[6 ]Roewer, L. (2013). DNA fingerprinting in forensics: Past, present,

future. Investigative Genetics, 4(1), 22. https://doi.org/10.1186/2041-
2223-4-22

[7] Cawood, A. H. (1989). DNA fingerprinting. Clinical Chemistry, 35(9),

1832–1837.

[8] Butler, J. M. (2015). Advanced topics in forensic DNA typing:

Interpretation. Elsevier.

[9]Mattei, A., & Zampa, F. (2023). Error rates and proficiency tests in the
fingerprint domain: A matter of perspective and conceptualization. Forensic Science
International, 348, 111651. https://doi.org/10.1016/j.forsciint.2023.111651