This presentation dives deep into the powerful world of DNA profiling and its essential role in modern forensic science. Beginning with the history of DNA fingerprinting, pioneered by Sir Alec Jeffreys in 1985, the presentation traces the evolution of forensic DNA analysis from the early days of RFLP (Restriction Fragment Length Polymorphism) to today's highly efficient STR (Short Tandem Repeat) typing methods. You will learn about the key steps involved in STR analysis, including DNA extraction, amplification using PCR, capillary electrophoresis, and allele interpretation using electropherograms (EPGs). Detailed slides explain how STR markers, classified by repeat unit length and structure, are analyzed for human identification with remarkable precision—even from minute or degraded biological samples. The presentation also introduces other DNA typing techniques such as Y-chromosome STR analysis, mitochondrial DNA (mtDNA) profiling, and SNP typing, alongside a comparative view of their strengths and limitations. Real-world forensic applications are explored, from crime scene investigations, missing persons identification, and disaster victim recovery, to paternity testing and cold case resolution. Ethical considerations are addressed, emphasizing the need for informed consent, privacy protections, and responsible DNA database management. Whether you're a forensic science student, a researcher, or simply curious about genetic identification methods, this presentation offers a comprehensive and clear overview of how STR typing works, its scientific basis, and its vital role in modern-day justice.

1. STR Analysis in forensics
By Muhammad Umar
Department of Biotechnology

2. Introduction
DNA profiling is the process of identifying individuals
using their unique genetic makeup.
Plays a crucial role in forensic science, especially in crime
scene investigations and legal proceedings.
Over the decades, DNA profiling has evolved from complex
and time-consuming methods to rapid and highly accurate
techniques.
This presentation will explore the origins, techniques,
classifications, and real-world uses of DNA profiling in
forensic science.

3. DNA, the fundamental blueprint of life, has become one of the most powerful tools in modern forensic
science. From solving cold cases to exonerating the innocent, the journey of DNA profiling has
reshaped justice systems worldwide.
forensic analysis does not examine the entire genome of individuals, but a much smaller number of
highly variable regions, comprising approximately one millionth of a genome.
It has also favored DNA sequences that are external to the genes, and are not believed to encode
biological traits.
Forensic techniques exploit those places in the genome, known as "polymorphic loci," which exhibit
detectable variations. These detectable variations (called “allelomorphs" or "alleles") can be of two
types: variation in the sequence of DNA bases; and length variation arising from differences in the
number of DNA bases between two defined end points.
PROGRESS OF DNA
PROFILING

4. The first legal application occurred In 1986. DNA analysis by Dr. Alec
Jeffreys cleared a falsely accused suspect in the rape and murder of 15-year-old
Dawn Ashworth. A mass screening of over 4,000 men followed, leading to the
arrest of Colin Pitchfork, who tried to avoid testing with a stand-in. His DNA
matched the crime scene, resulting in the first murder conviction using DNA
evidence in 1988. That same year, DNA was used in the U.S. to convict
Tommy Lee Andrews for rape.
'DNA fingerprinting' or DNA typing (profiling) as it is now known, was first
described in 1985 by an English geneticist named Alec Jeffreys. He found that
certain regions of DNA contained DNA sequences that were repeated over and
over again next to each other. He also discovered that the number of repeated
sections present in a sample could differ from individual to individual. By
developing a technique to examine the length variation of these DNA repeat
sequences, he created the ability to perform human identity tests.
Pioneers and Foundation
of DNA Profiling

5. Forensic DNA analysis has come a long way since its early beginnings in the 1980s, with the emergence of several different techniques. STR
analysis is now the primary method but, before its invention, restriction fragment length polymorphism (RFLP) was the dominant technique.
Additionally, since the adoption of STR approaches, other methods such as Y chromosome analysis, mitochondrial DNA (mtDNA) analysis,
single nucleotide polymorphism (SNP) typing and mini STR analysis have also been developed.
DNA fingerprinting technique began years ago with the introduction of restriction fragment length polymorphism (RFLP), and in the 1984, RFLP
method gave way to PCR methodologies(1985) that had the advantage of being able to amplify DNA. After several improvements and
refinements to the PCR-based tests, the forensic community came to an agreement on the use of STRs.
Types of forensic DNA analysis

6. RFLP was the first DNA typing method. DNA was typed using the RFLP method,
which comprised central elements of sequences made up of 30-100 repetitions. A
significant amount of intact genomic DNA is needed for DNA characterization using
the restriction fragment length polymorphism technique. As a result, the restriction
fragment length polymorphism technique was often inapplicable.
RLFP Technique
Multi-Locus
1. Fragment Separation by Gel Electrophoresis
The resulting DNA fragments (1–20 kb in size) are separated based on length using gel
electrophoresis.
2. Hybridization with Multi-locus Probes
Probes that bind to multiple minisatellite loci are applied, producing visible patterns Of
bands.
3. Pattern Analysis (DNA Profiling)
The unique banding pattern, originally called a "DNA fingerprint," is analyzed to identify
individuals — now termed DNA profiling.
4. DNA Extraction and Digestion
DNA is extracted and cut into fragments using restriction enzymes, targeting regions with
variable minisatellites.
Southern Blotting technique

7. VNTRs
Single-locus Probes
The next step in the development of forensic DNA work utilized the
same RFLP technology; however, the probes used to visualize the
product were altered to target only one locus at a time. These systems
were referred to as VNTRs.
• After restriction digestion, and run on gel, the DNA is transferred
onto a nylon membrane(southern Blotting) and radioactive probes are
added for visualization
• The alleles were characterized by a measurement of their molecular
weight. Each allele was an integer multiple of the repeat sequence
plus the flanking DNA(the DNA on either side of repeats).
While VNTR loci are still in use in some laboratories, they have largely
been replaced by STR loci.

8. PCR System
PCR was invented in 1986 by Kary Mullis at Cetus Corporation in
California.
the PCR technique uses an enzyme called a "polymerase" to replicate (or
"amplify") a DNA sequence 100-2,000 base pairs long.
PCR produces millions of copies of the initial DNA sequence through a
chain reaction in which the products of one round of replication become
templates for the next.
A PCR system can generate profiles from samples as small as 0.3-0.5
nanograms of DNA, about a hundred times smaller than required for
RFLP, and corresponding to the amount of DNA present in a few
hundred sperm cells or a blood spot the size of a large pin- head.

9. Forensic DNA markers are arbitrarily
plotted in relationship to four quadrants
defined by the power of discrimination for
the genetic system used and the speed at
which the analysis for that marker may be
performed.
Note that this diagram does not reflect the
usefulness of these markers in terms of
forensic cases.
COMPARISON OF DNA
TYPING METHODS

10. Short Tandem Repeats (STRs)
Eukaryotic genomes have repetitive DNA sequences. These DNA sequences are very small
in size and are usually determined by the length of the nucleus of each repetitive unit and the
number of repeats of the nucleus of each unit. Regions of DNA have repetitive units’ 2 to 6
bp microsatellite, or commonly called short tandem repeats (STRs).
Most STRs are found in the noncoding regions, while only
about 8% locate in the coding regions. accounting for about
3% of the entire genome.
On average, one STR occurs per 2,000 bp in the human
genome. The most common STRs in humans are A-rich
units: A, AC, AAAN, AAN, and AG.

11. On the basis of different repeat units, STRs can be classified into different types. On
the one hand, according to the length of the major repeat unit, STRs are classified into
mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats.
The total number of each type decreases as the size of the repeat unit increases. The
most common STRs in the human genome are dinucleotide repeats.
On the other hand, according to the repeat structure, STRs are classified into:
• Perfect repeats (simple repeats), containing only one repetitive unit
• Imperfect repeats (compound repeats), consisting of different composition repeats
The STR locus is named as, for example, D3S1266, where:
• D DNA
• 3 Chromosome 3
• S STR
• 1266 Unique Identifier
Types and Nomenclature of STR Markers

12. A DNA database is a government database of DNA profiles and/ or DNA samples (DNA Databank) which can be used
by law enforcement agencies to identify suspects of crimes. The first government database, the National DNA Database
(NDNAD) was set up by the United Kingdom in April 1995.
Beginning in 1996, the FBI Laboratory sponsored a community-wide forensic science effort
to establish core STR loci for inclusion within the national DNA database known as CODIS (Combined DNA Index
System). It began in 1997 by involving 17 candidate loci, but at the project meeting after a few weeks, 13 loci were
selected as standard for forensic analysis by STRs.
Despite not having its own DNA Database, Pakistan, In 2002, the Executive Committee of the National Economic
Council permitted the National Forensic Science Agency (NFSA) to function as an autonomous body. Since 2006, it
has been teaching, training and creating additional forensic labs all over the country. The Punjab Forensic Science
Agency (PFSA) in Lahore is equipped with state-of-the-art technology that is being used throughout the world. Despite
being a provincial agency, it also accommodates cases from other provinces. The NFSA also has its own CSU covering
Islamabad and nearby districts.
DNA database

13. The DNA fingerprinting process starts with collecting a
reference sample, typically using a buccal swab due to its
simplicity and low risk of infection. If unavailable, other
biological materials like blood, saliva, semen, or stored
tissues may be used. The reference sample is then analyzed
to create a DNA profile, which is compared with another
sample to check for a match. This technique plays a crucial
role in forensics, paternity testing, and identifying unknown
individuals. The techniques such as RFLP, STR, PCR,
mtDNA are applied.
DNA typing process

14. In order to perform analysis on STR markers, the invariant flanking regions surrounding the repeats must be
determined. Once the flanking sequences are known then PCR primers can be designed and the repeat region
amplified for analysis.
The following steps are taken for STR typing in the conventional order for DNA typing:
• DNA extraction is performed to decide the amount of DNA present
• amplify STR loci
• separate PCR amplicons on a genetic analyzer
• examine the resultant data using bioinformatics
• and compare the data from one sample to directories with previously generated short tandem repeats sets
Steps for STR Analysis

15. Capillary Electrophoresis
Its a technique used to separate ionic species based on
their charge and size. In this setup, a fused silica
capillary is filled with a background electrolyte. A
high voltage is applied between the anode (positive
electrode) and cathode (negative electrode), creating
an electric field that drives the movement of sample
ions from the injector toward the detector. Smaller or
more highly charged ions migrate faster through the
capillary, allowing for separation. The detector records
the separated components as they reach the end of the
capillary.

16. Interpretation of STR Results
Detection and Analysis Using EPG
An EPG (electropherogram) is a graph that visually represents DNA molecules as they exit a separation column. As
DNA molecules move through the column, a detector captures their exit and sends the data to a computer. The software
then converts this signal into an EPG, which analysts use to determine which alleles are present in a sample.
The Kit:
Manufactured kits provide the key chemicals to enable the amplification and analysis of DNA
extracted from a range of samples such as blood, hair, and stains. These are also called multiplexes.
Every graph has two axes, the vertical (y-axis) and
the horizontal (x-axis). In the EPG, the horizontal
axis generally shows the size of the DNA molecule,
which appears; smaller molecules will show as
peaks toward the left-hand side of the EPG and
larger molecules to the right.

17. Comparison of the DNA profiles for two
individuals obtained with multiple short
tandem repeat markers. STR length variation
at unique sites on 10 different chromosomes
are probed with this DNA test to provide a
random match probability of approximately 1
in 3 trillion.
A gender identification test also indicates that
the top sample is from a male while the bottom
sample is from a female individual. These
results were obtained from a spot of blood the
size of a pin head in less than five hours. The
DNA size range in base pairs is shown across
the top of the plot. Results from each DNA
marker are indicated by the letters A-J.

18. Mitochondrial
DNA (mtDNA)
mtDNA is found outside the nucleus in the
mitochondria and is passed from mother to
child.
Useful in analyzing hair shafts, bones, and
other old/degraded samples.
Less discriminating than nuclear DNA (same
mtDNA among maternal relatives).
Valuable for identifying missing persons or
historical remains.

19. Y-Chromosome Analysis
Focuses on short tandem repeats found
only on the Y chromosome.
Since the Y chromosome is inherited
along paternal lines, all male relatives
share the same Y-STR profile.
Useful in sexual assault cases with
multiple male contributors.
Cannot distinguish between brothers or
male paternal relatives.

20. Why STRs Are Preferred
• The development of PCR improved the sensitivity of the analysis. • The
time taken per analysis was reduced to less than 24 hours.
• The cost effectiveness of the method was greatly improved due to a
reduction in the labor required.
• The shorter STR loci allow the analysis of degraded DNA samples, which
are frequently encountered by forensic scientists. This was because these
short segments of DNA stood a higher chance of being intact after
degradation.
• STR loci can be multiplexed together using several different STR primer
pairs to amplify several loci in one reaction. Multiplexing was further
facilitated by the development of dye-labeled primers that could be
analyzed on automated DNA sequencers.
• The collection of data was automated, and the analysis of data was partially
automated.

21. Real-World Applications of STR Analysis
Crime Scene Investigations: Matching suspects to evidence.
1
Missing Persons Identification: Comparing DNA with relatives.
2
Disaster Victim Identification: Mass casualty events (e.g., earthquakes, plane crashes).
3
Paternity Testing: Legal and personal identity resolution.
4
Cold Case Resolutions: Revisiting unsolved crimes with preserved evidence.
5

22. Ethical
Considerations
Privacy: Concerns over DNA data storage and use.
Consent: Importance of informed consent for
collection.
Discrimination: Risk of genetic discrimination in
employment, insurance, etc.
Data Security: Safeguarding DNA databases from
misuse or breaches.
Ethical frameworks and legal regulations are
essential for responsible use.

23. DNA profiling has revolutionized forensic science and justice.
STR analysis remains the most widely used and trusted method.
Multiple techniques offer solutions for a wide range of forensic challenges.
With advancing technology, DNA profiling will become even more precise and
accessible.
Ethical and legal safeguards are essential for maintaining public trust.
Conclusion

24. THANK YOU!