DNA Barcoding and

Fingerprinting

Nesrin E. Maca-ampao
 Points to Discuss

01 02 03
Introduction DNA Barcoding DNA Fingerprinting
Molecular Science, the Basics Definition, Process, Definition, Different
and Key Terminologies Applications, Advantages and Techniques, Application,
Limitations Advantages and Limitations

04 05
Similarities and Differences Conclusion
Comparison of DNA Barcoding vs Summary and Future Outlook
DNA Fingerprinting
 Introduction
Molecular Science, the Basics and Key
Terminologies
 MOLECULAR SCIENCE AND
MOLECULAR BIOLOGY
● Molecular science has a significant impact on the field of
diagnostics and medicine, contributing to our understanding
of diseases, development of diagnostic tools, and
advancements in treatment.
● In the context of DNA barcoding and fingerprinting,
molecular science is applied to molecular biology, to classify
and identify species by analyzing a short, standardized region
of their DNA, and making a unique “fingerprint” of identity.
SHORT HISTORY OF MOLECULAR
BIOLOGY
● 1865: Gregor Mendel’s work on inheritance sets the stage for
understanding genetic principle
● 1869: Friedrich Miescher isolates “nuclein,” the substance we now know
as DNA.
● 1953: James Watson and Francis Crick propose the double helical
structure of DNA.
● 1958: Francis Crick’s “central dogma” outlines the flow of genetic
information.
SHORT HISTORY OF MOLECULAR
● 1961: François Jacob andBIOLOGY
Jacques Monod describe the operon model.
● 1970: Hamilton Smith and Daniel Nathans develop the first DNA-
cutting enzyme, a key tool for genetic engineering.
● 1977: Frederick Sanger’s development of DNA sequencing techniques
revolutionizes molecular biology.
● 1983: Kary Mullis invents the polymerase chain reaction (PCR) for
DNA amplification.
● 1990: The Human Genome Project begins, aiming to sequence the entire
human genome.
 DEFINITION OF SOME TERMS
DNA (Deoxyribonucleic Acid)
Genetic material found in cells that carries the instructions for the development,
functioning, growth, and reproduction of all known living organisms. It has a double-
helix structure made up of nucleotides.
RNA (Ribonucleic Acid)
A molecule similar to DNA but typically single-stranded. It plays various roles in gene
expression, including transferring genetic information from DNA to protein synthesis.
Gene
A gene is a segment of DNA that contains the instructions for making a specific protein
or performing a particular function in an organism. Genes are the basic units of
heredity
Genome
Complete set of an organism’s genetic material, including all of its genes and non-
coding sequences of DNA
DNA
Barcoding
Definition, Process, Applications, Advantages
and Limitations
“Everyone has a
barcode”
DNA barcoding is a molecular
technique that seeks to identify
species by comparing a
standardized DNA region, typically
the COI gene in animals and a
region of the rbcL and matK genes
in plants.
 THE CO1 / COX1 GENE
● Also known as Cytochrome c Oxidase Subunit I, is a critical gene found
in the eukaryotic organisms, including animals
● The CO1 gene is typically located in the mitochondrial genome, a
separate set of genetic material distinct from the nuclear DNA.
● It plays a crucial role in the respiratory chain and energy production
within cells.
● The CO1 gene is highly conserved within species but can exhibit
variability between different species. This variation is particularly useful
in DNA barcoding, a technique used to identify and classify species
based on differences in the CO1 gene sequence.
WHY IS THE CO1 / COX1 GENE USED?
● Essential and Present in all eukaryotic cells (CO1 gene is available in all
eukaryotes with a mitochondria because it is important for cellular
respiration)
● Each cell has identical copies (CO1 gene is almost always identical in
any of the cells in the organism, so most tissue samples can be used)
● Close, but not too close (CO1 gene is highly conserved in each species
that it is the same for one species but different from another)
● There is an exception, however! Animals benefit more from CO1. Plants
have slower change in CO1 over generations which means different
plant species may have the same CO1
● Plants therefore uses rbcL or matK gene
 The process of DNA Barcoding

SPECIMEN DNA
SAMPLING AMPLIFICATION

DNA DNA
EXTRACTION SEQUENCING
 SPECIMEN SAMPLING
Most tissues from an organism can be used for specimen sampling, but fresh
and soft tissues are easier to process

However, it is not always possible to immediately process the organism. In this

case, preservation by freezing, drying or chemical fixation by formalin or
ethanol is used to prevent DNA degradation

Samples are collected as “aseptically” and as “sterile” as possible. This is to

prevent cross-contamination and as a result have a wrong identification of the
organism (example being commonly human contamination)
 DNA EXTRACTION
DNA Extraction sounds as it is, to Extract the DNA in the specimen for testing.
Molbio laboratories are extensive and have multiple steps. Extraction kits
from companies are available in the market.

However, it usually involves these few steps: Homogenization, Cell Lysis,

Protein Removal, DNA Precipitation and Rehydration of Buffer

In homogenization, the tissue is “mixed uniformly” by manually destroying the

tissue to become “loose cells”. For plant tissues, it is made usually into a powder,
and for animal tissues it is made into a paste or a slurry.
 DNA EXTRACTION
Once it is homogenized, the next step is to break down individual cells: to
LYSE them by destroying their cell membrane, usually with detergents and
enzymes. This makes the individual cells burst and release the DNA along with
other materials like protein.

DNA is needed, not the proteins. Proteins will interfere with DNA analysis so
the next step is to remove them from the suspension. This is done by adding
protein precipitation and enzyme treatments.

After removing the proteins, the DNA is still distributed within the suspension.
The next process is to “concentrate” the DNA by precipitation. This is usually
done by adding ethanol. Ethanol clumps up and precipitates the DNA forming
solid white strings. This is centrifuged, and the DNA is washed. A buffer is then
added to make it a liquid suspension again with pure DNA, and is ready for the
next step.
EXPERIMENT: EXTRACTING
YOUR OWN DNA
MATERIALS:
• WARM WATER
• SALT
• DISHWASHING LIQUID
• 95% ISOPROPYL ALCOHOL
 PROCEDURE:
• Add a tablespoon of salt to two cups of warm
water
• Gargle ¼ cup of the saltwater solution for 1
min and spit it out of a glass
• Add two drops of dishwashing liquid and stir
gently
 PROCEDURE:
• Add ½ cup of 70 percent ethyl or isopropyl
alcohol to the saltwater gargle + dishwashing
solution
• Wait for 3 mins until white strands form
• Use a stick to pull the white strings out. That
is DNA!
•
RATIONALE:
The saltwater gargle solution will harvest
cheek cells from the mouth of the participant.
Both the salt from the saltwater and the added
dishwashing liquid acts as a cell lysis agent
and protein removal agent. Alcohol
precipitates the DNA from the mixture,
forming white strands.
 VIDEO:
• https://youtu.be/fQo4bqV29Gs?si
=9DPBnfoQP1AT1fP-
 DNA AMPLIFICATION
After extraction of DNA, you are left with a Purified DNA suspended in a
buffer. However, the DNA collected is still small enough to be analyzed in the
laboratory. This is where DNA Amplification comes in use.

DNA amplification is the process of making multiple copies of a specific DNA

sequence. It is essential in DNA barcoding to ensure there is enough DNA
material for analysis.

In short, the idea of DNA amplification is to “artificially” make copies of the

purified extracted DNA. This is commonly done with what is known as the PCR
technique.
POLYMERASE CHAIN REACTION
● Also known as PCR, is a laboratory technique that enables the selective
amplification of a particular DNA segment
● It creates “more copies” of a DNA segment (gene) for it to be abundant
enough to be analyzed and sequenced (for the later part of the DNA
barcoding process)
● In this case, the amplified gene is the CO1 gene for animals and
rbcL/matK gene for plants
● It involves three main steps: denaturation, annealing, and extension,
which are repeated in a cycle to exponentially increase the DNA copies.
 WHAT IS NEEDED FOR PCR?
DNA Template
The DNA to be amplified, which contains the target region of interest (e.g., the barcode
region). This is the Extracted DNA
Primers
Short DNA sequences that bind specifically to the regions flanking the target DNA,
defining the region to be amplified.
DNA Polymerase
An enzyme, such as Taq polymerase, that synthesizes new DNA strands by extending
from the primers
Nucleotides
Building blocks (A, T, C, G) used to create new DNA strands.
Buffer Solution
Provides the necessary pH and salt conditions for the reaction.
Thermocycler
A machine that controls the temperature changes required for each PCR cycle.
THERMOCYCLER/
THERMAL CYCLER
 HOW DOES PCR WORK?
● There are three main steps in PCR: Denaturing, Annealing and
Extension.
● Denaturation: The PCR reaction begins with high heat, which
separates the double-stranded DNA into two single strands. This
opens the DNA for “copying”
● Annealing: The temperature is lowered, allowing the primers to bind
to their complementary sequences on the DNA template.
● Extension: The temperature is raised again, and DNA polymerase
synthesizes new DNA strands complementary to the template,
extending first from the primers, using the free nucleotides (C to G,
A to T)
 HOW DOES PCR WORK?
● As observed, this process is almost the same as DNA Duplication in
cells, but in this process, only a part of the DNA (the barcode) is
needed to be copied for analyzing. It is also “artificial ” in nature and
controlled by the thermocycler.
● These three steps (denaturation, annealing, extension) are repeated in
a thermal cycler for multiple cycles. With each cycle, the target DNA
region is doubled, resulting in exponential amplification.
● After several cycles, there are millions of copies of the target DNA
segment, making it suitable for further analysis, such as sequencing
or species identification.
 Video:
● https://youtu.be/a5jmdh9AnS4?si=YzqOtFF8vruJkReD
 DNA SEQUENCING
After many of the DNA fragments have been amplified (CO1 and rbcL/matK),
it is now brought to the complex step to determine the genome of the
organism, the DNA Sequencing

DNA sequencing is a critical step in DNA barcoding, as it reveals the precise

sequence of the chosen barcode region. This sequence data is essential for
species identification and cataloging in biodiversity research.

There are two main techniques for DNA Sequencing. The Sanger Method which
is the more traditional method and the Next Generation Sequencing Method
which is more faster and efficient.
HOW DOES DNA SEQUENCING WORK
Sanger sequencing, also known as chain termination sequencing, is the
traditional method used in DNA barcoding for sequencing short DNA
fragments.

On the other hand, Next generation sequencing is a newer technology which

has the same concept as Sanger sequencing. However, Unlike traditional
Sanger sequencing, which sequences DNA one strand at a time, NGS
techniques parallelize the process, enabling the simultaneous sequencing of
millions to billions of DNA fragments.
 DNA SEQUENCING PROCESS
• Denaturation: The DNA sample is heated to separate the double-stranded
DNA into single strands.

• Primer Annealing: A sequencing primer (complementary to the DNA

template) is added, allowing DNA polymerase to start synthesizing a new
strand of DNA.

• DNA Polymerization: DNA polymerase incorporates labeled nucleotides

(A, T, C, G) into the growing DNA strand.

• Termination: Each labeled nucleotide is modified to prevent further

extension, resulting in a collection of DNA fragments of varying lengths,
each ending with a specific labeled base.
 DNA SEQUENCING PROCESS

• Electrophoresis: The labeled DNA fragments are separated by size using

electrophoresis, typically in a gel matrix or capillary tube, depending on
the sequencing platform.

• Detection: As the DNA fragments move through the gel or capillary, they
pass a detector that records the position and fluorescence of each labeled
fragment.
 EASIER VISUALIZATION
For simplicity, DNA Sequencing is like DNA Amplification as well. The only
difference is that the nucleotides (CGAT) are labelled while the DNA is
extending, mostly with fluorescence and color. This is detected by the machine
to determine what nucleotide is paired

For example, if a gene is CGAT then the process will pair it with GCTA. If G
fluorescence green, C fluorescence blue, T fluorescence red and A fluorescence
yellow, it will show green, then blue, then red then yellow

This will be detected as G then C then T then A pairing with the DNA that is
unknown. This means that the Unknown DNA genome is C G A T
 DNA SEQUENCING PROCESS
• Data Analysis: The raw data from the sequencer is processed to generate a
chromatogram, which represents the sequence of the DNA template.
Bioinformatics software is used to analyze the chromatogram and convert
it into a digital DNA sequence.

• Quality Control: Quality control measures are applied to ensure the

accuracy and reliability of the sequencing data. This includes checking for
base-calling errors and assessing the sequence’s overall quality.

• Species Identification: The obtained DNA sequence from the barcode

region is compared to reference sequences in online databases, such as
GenBank or BOLD (Barcode of Life Data Systems), to identify the species
of the organism.
 Video:
https://youtu.be/1xU1oNB46YI?si=69yVnNdyP6eXDtjK
 APPLICATIONS IN MOLECULAR
BIOLOGY
Species Identification
It’s used for accurate and rapid identification of species, including endangered or cryptic ones,
aiding conservation efforts. It can also identify pathogens, which also includes bacteria and
viruses (though CO1 or any eukaryotic genes are not used since they are prokaryotic)

Biodiversity
DNA barcoding helps identify and catalog species in ecosystems, providing insights into
species diversity and distribution.

Food Identification
DNA barcoding helps identify and catalog species in ecosystems, providing insights into
species diversity and distribution.
ADVANTAGES LIMITATIONS

• Many Applications in • Limited Reference

the field of Molecular Database
Biology • Genetic Variations
• Used for • Hybrid Species
identification and • DNA Degradation and
conservation Cross contaminations
• Rapid and Accurate • Ethical and Privacy
• Applicability Across Concerns
Taxa
DNA Fingerprinting
Definition, Different Techniques , Applications,
Advantages and Limitations
“A fingerprint will
show who you are”

DNA fingerprinting, also known as DNA

profiling or DNA typing, is a forensic
technique that identifies individuals based on
their unique DNA patterns.
 GENETIC LOCI
● Each person’s DNA contains regions with repetitive sequences known as
Variable Number Tandem Repeats (VNTRs) or Short Tandem Repeats
(STRs). These regions vary in length among individuals, creating unique
DNA profiles.
● DNA Barcoding uses CO1 Gene or any other gene that is present and the
same for the same species. DNA Fingerprinting identifies this unique
genetic makeup which is different for every individual of the same
species.
 DIFFERENT TECHINIQUES

STR Analysis PCR RFLP

• The discussion for the process of DNA Fingerprinting will be
very short because the same process and techniques used in DNA
Barcoding are used in DNA Fingerprinting as well. It has a lot of
similarities that it almost are the same and just called through a
different name. Most difference of DNA Barcoding and
Fingerprinting are based on their applications, which will be
discussed later.
RESTRICTION FRAGMENT LENGTH
POLYMORPHISM
RFLP is a molecular biology technique that detects variations in DNA
sequences by analyzing the lengths of DNA fragments produced after digestion
with restriction enzymes.

RFLP relies on the fact that DNA sequences vary between individuals,
resulting in differences in the locations recognized by restriction enzymes.
When DNA is digested with a specific enzyme, it produces fragments of
varying lengths based on the presence or absence of restriction sites.

The technique is very similar to DNA amplification, just more specific DNA
sequences that can identify an individual
 RFLP PROCESS
• DNA is extracted and purified from a sample.

• The DNA is digested with one or more restriction enzymes that recognize
specific DNA sequences.

• The resulting DNA fragments are separated by size using gel

electrophoresis

• A DNA probe, typically a radioactive or fluorescently labeled fragment, is

used to hybridize with the separated DNA fragments.

• The location and length of the labeled fragments are visualized to create a
DNA fingerprint.
SHORT TANDEM REPEAT ANALYSIS

STR analysis is a molecular biology technique used to analyze specific regions

of a DNA sample. These regions contain short sequences of DNA (2 to 6 base
pairs) that are repeated consecutively.

Each individual has a unique number of repeats at these STR loci. By

analyzing the number of repeats at several STR loci, a unique DNA profile or
fingerprint for an individual can be generated.
 STR ANALYSIS PROCESS
• DNA is extracted from a sample, such as blood or hair.

• PCR (Polymerase Chain Reaction) is used to selectively amplify the STR

regions of interest.

• The amplified DNA fragments are then separated by size using capillary
electrophoresis.

• The resulting data, showing the lengths of the STR fragments, is used to
create a DNA profile.
 APPLICATIONS IN MOLECULAR
BIOLOGY
Forensic Identification
DNA fingerprinting is widely used in solving crimes by matching DNA from crime scenes to
suspects or existing DNA databases. It also helps identify victims of disasters or crimes
when traditional identification methods are not possible.

Relationship Testing
It is employed to establish biological relationships, including paternity, maternity, and
sibling relationships.

Historical and Anthropological Research

DNA fingerprinting contributes to genealogical research and understanding human migration
patterns and genetic diversity.
ADVANTAGES LIMITATIONS

• High Precision • Costly and Time

• Permanent and Consuming
Unchanging • Needs a high quality
• Non-invasive specimen
• Applicable for Legal • Environmental factors
Proceedings that may interfere
• False positives and
False negatives
SIMILARITIES AND
DIFFERENCES
 COMPARISON OF DNA
BARCODING FINGERPRINTING
Both DNA barcoding and DNA fingerprinting are used for genetic identification.

Identifies and classifies species using Identifies individuals within a species using
specific genes. multiple genetic loci, primarily in humans.

Applies to various species and is helpful for Primarily used for human identification and
differentiating species. genetic profiling within a species.

Focuses on specific, standardized genetic Analyzes multiple genetic loci, often

regions (e.g., CO1, rbcL) that are conserved including STRs or VNTRs, which can be
within species but vary between species. highly variable among individuals.
 DNA BARCODING
LABELS WHO YOU ARE

DNA FINGERPRINTING
TRACKS WHO YOU ARE

TOGETHER, THEY
IDENTIFY WHO YOU ARE
CONCLUSION
SUMMARY AND FUTURE OUTLOOK
• DNA Barcoding is used for species identification in diverse organisms.
• It targets specific standardized genetic regions.
• The process include DNA Extraction, Amplification and Sequencing
• Applies to biodiversity research and species differentiation.
• DNA Fingerprinting is used for individual identification within a species
primarily in humans
• It analyzes multiple genetic loci, often STRs or VNTRs.
• Techniques include RFLP and STR Analysis
• Mainly applied in forensic science, paternity testing, and human genetics.
• Both techniques involve genetic analysis for identification, but DNA barcoding
is geared toward species differentiation, while DNA fingerprinting focuses on
individual identification within a species.
THANK YOU! ^^