DNA barcoding is a standardized method to identify species using a short genetic marker from a standardized portion of the genome. It involves building a reference library of DNA barcodes from identified specimens of known species. Unknown samples can then be identified by comparing their barcodes to sequences in the reference library. The standard barcode region for animals is the COI gene from mitochondrial DNA. DNA barcoding has many applications, including identifying species across all life stages, identifying fragments or processed products, tracking disease vectors, distinguishing cryptic species, and detecting illegal wildlife trade. It provides an alternative identification method that can complement morphological identification.

2. DNA Barcoding
In Animals

3. CONTENTS
• HISTORY• INTRODUCTION
• BARCODE OF
LIFE
COMMUNITY
• HOW
BARCODING
IS DONE
• WHY
BARCODING
IS DONE
• APPLICATIONS
• DNA BARCODING
IN PAKISTAN
• BARCODE DATA ANALYSIS
• CONCLUSION

4. I. Taxonomic Identification
• Morphological characters
• Breeding
• Biology
• Host plants etc.
II. Molecular Identification
 RNA/DNA nucleotide data
 Protein analysis
Biological Unit Identification

5. Molecular Identification
Use of DNA/RNA and proteins sequence data for
individual or species identification by
• Sequence comparisons
• Constructing phylogenetic trees
• Other available tools

6. Why Molecular Identification?
• Clear genetic basis
• Deformed/broken samples are OK
• DNA from fossilized specimens can be used
• Identification of immature stages, or concealed
stages possible
• Quick and unbiased
• Reliable

7. Deoxyribonucleic Acid
• DNA is:
• The hereditary material in humans
and almost all other organisms.
• Same in nearly every cell of body
• Mostly located in the cell nucleus
(nuclear DNA)
• Also found in small amount in the
mitochondria (mitochondrial DNA).
ghr.nlm.nih.gov

8. Barcodes:
• Uniform Product Code
• Consists of hidden language
made up of series vertical bars
lines of varying width
• Used in identification by optical
or laser scanner
The system of showing information
in the form of barcodes
Barcoding
en.wikipedia.org/wiki/Barcode

9. DNA Barcoding
• DNA barcoding is a standardized approach to identifying
plants and animals by minimal sequences of DNA, called
DNA barcodes.
• A DNA barcode is a short gene sequence taken from
standardized portions of the genome, used to identify species.
www.barcodeoflife.org/content/about/what-dna-barcoding

10. Marker
• The standard region
used to generate DNA
barcode
• Different in different
species
• In animals it is COI or
cox1 (cytochrome C
oxidase 1) present in
mitochondrial gene
www.dnabarcodes.org

11. An Internal ID System
The Mitochondrial Genome

12. The Chosen Gene

13. Why COI Gene?
• Standard region
• Lack insertions or deletions
• Ease of isolation
• Greater differences among
species
• High copy number (100-10,000 )
• Relatively few differences within
species
• Absence of introns
• Range of mutational rates in
different regions of the molecule

14. Standard Barcode region for Prokaryote
• SSU • lSU
Nuclear DNA - rRNA• Easily available
• High copy number
• High degree of
variation
• Find and Amplify
• Inter Transcribed
spacer
• Ribosomal genes code for rRNA
• Spacer regions are transcribed but then removed
• Region has restriction site polymorphism between
species

15. Using DNA Barcodes
• Establish reference library of barcodes from identified voucher
specimens
• If necessary, revise species limits
• Then:
• Identify unknowns by searching against reference
sequences
• Look for matches (mismatches) against ‘library on a chip’
• Before long: Analyze relative abundance in multi-species
samples
www.springer.com/us/book/9781617795909

16. History Of DNA Barcoding
• Carl Woese gave this concept
first time.
• He used rRNA and molecular
markers like rDNA and
mtDNA to discover Archea i.e.
prokaryotes and then for
drawing evolutionary tree.
• In 2003, Paul Hebert,
researcher at the University of
Guelph in Ontario, Canada,
proposed “DNA barcoding” as
a way to identify species.
Paul Hebert
www.barcodeoflife.org/content/about/what-dna-barcoding

17. Our Planet:
Home to approximately
10-100 million species
Our Brain:
Able to recall and
recognize perhaps 1000
species
Why DNA Barcoding?

19. Ten Reasons For Identifying Species By
DNA Barcodes
1. Works with fragments
2. Works for all stages of life
3. Unmasks look-alikes
4. Reduces ambiguity
5. Makes expertise go further
6. Democratizes access
7. Opens the way for an electronic handheld field guide, the Life
Barcode
8. Sprouts new leaves on the tree of life
9. Demonstrates value of collections
10. Speeds writing the encyclopaedia of life

20. • Promote barcoding as
a global standard
• Build participation
• Working Groups
• BARCODE standard
• International
Conferences
• Increase production of
public BARCODE
records
Networks, Projects, Organizations
Barcode of Life Community
http://www.barcodeoflife.org/content/about/barcoding-landscape

21. There are four components of
barcoding projects:
1. The Specimen Collection
2. The Laboratory Analysis
3. The Data Base
4. The Data Analysis
Components Of Barcoding Projects
http://www.barcodeoflife.org/content/about/what-dna-barcoding

22. Method
http://www.barcodeoflife.org/content/about/what-dna-barcoding

24. DNA Barcoding Lab Scenes

30. Barcode Analysis
• Nucleotide
identities/matches
• Distance analysis
• Barcoding gap
• Cluster analysis

31. Methods

33. Identification Of All Life Stages
www.springer.com/us/book/9781617795909

34. Identification Of Fragments Or
Products Of Organisms
• Illegally traded bushmeat, sharkfins, skins

35. ?
Food Safety and Accuracy

36. For Example

37. Disease Vectors
Culex pipiens
WEST NILE VIRUS
Culicens incidens
NON-VECTOR
Egg raft
?
?

38. Dengue transmission cycle West Nile Virus transmission cycle

39. Differentiating Cryptic Species
• Astraptes fulgerator, skipper
butterfly.
• Wide-ranging; southern U.S. to
northern Argentina.
• In northwestern Costa Rica,
comprises complex of 10 sympatric
species that are distinct in DNA
sequence (COI), larval coloration,
food plants, and subtle
morphological traits.

40. Sympatric larvae of Astraptes
fulgerator
Food plant: Celtis
iguanaea
Food plant:
Trigonia (2
species); larvae
will starve if
reared on plants
used by other
larval types.

41. Sisterspecies vs congeners
Panthera leo (lejon)
Panthera tigris (tiger)
Motacilla flava (gulärla)
Motacilla alba (sädesärla)
Carabus nitens (guldlöpare) Carabus coriaceus (läderlöpare)
Sisterspecies vs congeners
Sylvia minula (ökenärtsångare)
Sylvia curucca (ärtsångare)
Sisterspecies vs congeners
Pipistrellus pipistrellus (Pipistrell)
Pipistrellus pygmaeus (dvärgfladdermus)

42. Identification of stomach contents,
trace ecological food-chains

43. Other Applications
• Customs control
• Invasive species control
• Police
• Agriculture
• Forestry
• Conservation
• Education
• Estimating species diversity
• Resolving commercial disputes
• Etc
www.springer.com/us/book/9781617795909 www.ijergs.org

44. iBOL nodes

45. Canadian Centre for DNA
Barcoding
NIBGE
Funded by:
HEC ; iBOL
Pakistan Form Barcode Focal Point

46. Barcoding Species From
Pakistan
http://ibol.org/pakistan-to-form-barcode-focal-point/
• About 2,000 arthropod species have been barcoded under the
NIBGE-BIO collaborative project "sequencing DNA barcodes
of economically important insect species of Pakistan.“
• DNA barcoding is also being done on spiders, fishes and
mosquitos by different universities and researchers to identify
their species.

47. Illegal Wildlife Trade
• Pakistan is using DNA barcoding to check illegal wild life trade.
CASE STUDIES:
• Last year, over 200 black pond turtles destined for Bangkok,
were confiscated at the Karachi airport.
• Another consignment of turtles of the same species was caught at
the Chinese border which was then repatriated to Pakistan.
http://www.dawn.com/news/1203605

48. • The DNA testing method was first
tried in March 2015 when customs
officials confiscated a shipment of
nearly 2,000 kilograms of
freshwater turtle meat worth $60
million.
• The confiscated consignment was
of turtles native to the Indus River
and listed in the (CITES), that
restricts trade to ensure its survival.
• As many as 4,000 turtles were
killed and were being shipped out
of Pakistan.
Continued…

49. Strength
• Offers alternative taxonomic identification tool for situations
in which morphology is inconclusive.
• DNA barcoding can speed up identification of new species
• Potential capacity for high throughput and processing large
numbers of samples
• Identify specimens – a global identification system
• Discover new species – aid and speed up the discovery of the
remaining biodiversityFocus on one or a small number of
genes provides greater efficiency of effort.
• Cost of DNA sequencing is dropping rapidly due to technical
advances.
• Once reference database is established, can be applied by non-
specialist.

51. Thank
you!