DNA Barcoding
Kandhan. S,
M. Tech (Biotechnology)
PSG College of Technology

Barcodes
• Consists of hidden language made up of series
vertical bars lines of varying width
• Used in identification by optical or laser scanner
http://www.barcodesinc.com/generator/index.php
Aztec code
Cronto Sign
Digital matrix
EZ code
Nexcode
High capacity
color code
Data matrix
Maxi code
PDF 417
SPARQ Code
Qode
QR Code
Shot code

What is this ?
 DNA barcoding is a standardized approach
to identifying plants and animals by minimal
sequences of DNA, called DNA barcodes.
 DNA barcode - short gene sequences taken
from a standardized portion of the genome
that is used to identify species
DNA Barcoding

How it all started in 2003
Propose a CO1-based (~650bp of the 5’ end)
global identification system of animals,
and show the success (96.4-100%) of assigning
test specimens to the correct phyla, order and species
(Lepidoptera from Guelph) through a CO1-profile.
98% of congeneric species in 11
animal phyla showed
>2% sequence divergence in CO1

Banbury Center, Cold Spring Harbor
March 2003, September 2003

Proc Royal Soc London B 2003

http:www.barcoding.Si.edu

BIG challenge: 1.9M species
1 square = 10,000 species
Other plants

Collection and
Databasing
Central Nodes
Developing Nodes
Regional Nodes
Curation and
Identification
Sequencing Mirrored
Databases
Data Analysis
and Access
ICI is an alliance of researchers and biodiversity organisations in 21 nations.
All nations active in specimen assembly, curation and data analysis.
Sequencing and informatics support by regional and central nodes.

CBOL Member Organizations: 2009
• 200+ Member organizations, 50 countries
• 35+ Member organizations from 20+ developing countries

WHERE I’M
Nucleus

Standard DNA barcode for animals
Animal Cell
Mitochondrion
DNA
mtDNA
D-Loop
ND5
H-strand
ND4
ND4L
ND3
COIII
L-strand
ND6
ND2
ND1
COII
Small ribosomal RNA
ATPase subunit 8
ATPase subunit 6
Cytochrome b
COICOI
The Mitochondrial Genome
 5’ cytochrome c oxidase subunit I
 distinguishes 95% species
(648 bp)
15,000 Base pair
Herbert et al,2003

Why COI ?
 standard region
 lack insertions or deletions
 Protein closely-related species.
 Greater differences among species
 Copy number. (100-10,000 )
 Relatively few differences within species
 Absence of Introns Herbert et al,2003

Barcode regions of plant
Nuclear DNA
ITS Plastid DNA loci
Discrimination
Universality
Robustness
Plant Cell
Mat K
rbc L
trnH-psbA
atpF-F
psb k1
rpo C1
rpo B
rpo C2
ndh J
trn L
ycf 5
acc D
100,000 Base pair

• Discrimination
Barcoding regions must be different for each species. Ideally
you are looking for a single DNA locus which differs in each
species.
• Universality
Since barcoding protocols (typically) amplify a region of DNA
by PCR, you need primers that will amplify consistently.
• Robustness
Since barcoding protocols (typically) amplify a region of DNA
by PCR, also need to select a locus that amplifies reliably, and
sequences well.

% species discriminated
• ITS: 90.5%
• psbA-trnH: 60%
• matK: 33.3%
• ndhJ: 37.1%
• rpoB: 9.9%
• rpoC1:9.9%
• accD: 6.05 %
Nuclear non-coding
Plastid non-coding
Plastid coding
• accD, rpoB, rpoC1: variation too low for use as a single barcode
• matK and ndhF: more variable but with great variation of rate among
subgenera
• Non-coding regions (ITS and psbA-trnH spacer) performed better, but
required great manual effort for indel alignment

Based on recommendations by a barcoding consortium (Consortium for
the Barcode of Life, plant working group) the chloroplast genes rbcL and
matK universal plant barcodes.
– rbcL – chloroplast ribulose-1,5-bisphosphate carboxylate
– matK – chloroplast maturase K
Ratnasingham and Herbert, 2007
Why not COI
 Sequence divergent
Incorporation of forgein genes
Frequent transfer of some gene to Nucler gene0
Then plastid
Short
Easily alienable
Easily recoverable from even herbarium sample
Maternal interitence
mat K
rbc L

Comparison of Plant Barcode region

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
Kress et al,2007 Chase et al ,2005Conrad L. schock at al , 2012

Why Barcoding?
1)Works with fragments
2) Works for all stages of life
3)Unmasks look-alikes
4) Reduce ambiguity

5) Expertise to go further
6)Democratize access
7)Opens the way for an electronic handheld field
guide, the life barcoder
8)Sprouts new leaves on the tree of life
9) Demonstrates the value of collection
10) Speed writing the life of encylcopedia(http://eol.org/)

How the DNA Barcoding done
Step Involved in it
Sample collection & recording

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

Sample
collection
Biogeography classification
Expert
Taxonomist
•Museum
•Botanical garden
• Herbarium preparation
Wet lab Dry lab

DNA extraction, amplification & Sequencing
Amplification
Sequencing
Doyle and Doyle ,1998
Sanger, F. & Coulson, AR (1975)
Mullis et al ,1985

Sequence Align
UPLOAD IN BOLD AND
OTHER DATABASE
CONVERT TO
BARCODE

http://biorad-ads.com/DNABarcodeWeb/
Bio-rad barcode generator

Program behind DNA Barcode generator
• Luca &Howell
• Python 2.5 to 2.6
• shell window

Hollingworth,2008

Current Norm: High throughput
Large labs, hundreds of samples per day
ABI 3100 capillary
automated sequencer
Large capacity PCR and
sequencing reactions

Emerging Norm: Table-top Labs
Faster, more portable: Hundreds of samples per hour
Integrated DNA microchips Table-top microfluidic systems

Future in 20??
• Data in seconds to
minutes
• Pennies per sample
• Link to reference
database
• A taxonomic GPS
• Usable by non-
specialists

Advantage Of DNA barcoding
• Protection of Endangered Species ( Conservation)
• Tracking adulterations
• Identifying Agricultural pest
• Water quality testing
• Identification of all life stages, eggs, larvae, nymphs, pupa, adults
• Identification of fragments or products of organisms
• Identification of stomach contents, trace ecological food-chains
• Food control
• Customs control
• Invasive species control
• Disease vector control
• Police
• Agriculture
• Forestry
• Education
• Etc

Strength VS Weakness
• Alternative taxonomic
Identification tool
• Identification of new
species
• Work for all life stages
• Reveal undescribed
species
• No universal DNA
barcode region
• Difficult to resolve
recently diverged
species
• Identifies Inter-specific
genetic variation only
• Single approach

Conclusion
DNA barcoding has emerged and established
itself as a important tool for species-
identification and phylogenetics studies
it has proved useful in protecting Endangered
species, identifying agricultural pests and
disease vectors, tracking adulteration in
products and sustaining environment

Case studies

Hebert et al,2007

R.Sriama and Uma Shaanker,

Bha

Case studies

CONSERVE OUR ECOSYSTEM
This is where we stand today!

Why are u waiting for
Come out and play with DNA Bar-coding
to conserve the environment

References
• Smith, A., D.H. Janzen and P.D.N. Hebert. 2006. DNA barcodes reveal cryptic host-spceificity within the presumed
polyphagous members of a genus of parasitoid flies (Diptera: Tachinidae). Proc. Natl. Acad. Sci. USA 103: 3657-3662.
• Hajibabaei, M., D.H. Janzen, J.M. Burns, W. Hallwachs and P.D.N. Hebert. 2006. DNA barcodes distinguish species of tropical
Lepidoptera. Proc. Nat. Acad. Sci. USA: 103: 968-971.
• Ward, R.D., T.S. Zemlak, B.H. Innes, P.R. Last and P.D.N. Hebert. 2005. DNA barcoding Australia 's fish species. Phil. Trans. R.
Soc. Lond. 360: 1847-1857.
• Hebert, P.D.N. and T.R. Gregory. 2005. The promise of DNA barcoding for taxonomy. System. Biol. 54: 852-859.
• Barrett, R.D.H. and P.D.N. Hebert. 2005. Identifying spiders through DNA barcodes. Can. J. Zool. 83: 481-491.
• Lambert, D.M., A. Baker, L. Huynen, O. Haddrath, P.D.N. Hebert and C.D. Millar. 2005. Is a large-scale DNA-based inventory of
ancient life possible? J. Heredity: 96: 1-6.
• Hebert, P.D.N., M.Y. Stoeckle, T.S. Zemlak and C.M. Francis. 2004. Identification of birds through DNA barcodes. PLoS Biology
2: 1657-1663.
• Hebert, P.D.N., E.H. Penton, J. Burns, D.J. Janzen and W. Hallwachs. 2004. Ten species in one: DNA barcoding reveals cryptic
species in the neotropical skipper butterfly, Astraptes fulgerator . Proc. Natl. Acad. Sci. USA: 101: 14812-14817.
• Hebert, P.D.N., A. Cywinska, S.L. Ball and J.R. deWaard. 2003. Biological identifications through DNA barcodes. Proc. Roy. Soc.
Lond. Ser. B: 270: 313-321.
• Hebert, P.D.N., J.D.S. Witt and S.J. Adamowicz. 2003. Phylogeographic patterning in Daphnia ambigua: regional divergence
and intercontinental cohesion. Limnol. Oceanograph. 48: 261-268.

• Witt, J.D.S., D.W. Blinn and P.D.N. Hebert. 2003. The recent evolutionary origin of the phenotypically novel
amphipod, Hyalella montezuma offers an ecological explanation for morphological stasis in a closely allied
species complex. Mol. Ecol. 12: 405-413.
• Derry, A.M., P.D.N. Hebert and E.E. Prepas. 2003. Evolution of rotifers in saline and subsaline lakes: a
molecular phylogenetic approach. Limnol. Oceanograph. 48: 675-685.
• Gregory, T.R. and P.D.N. Hebert. 2002. Genome-size estimates for some oligochaete annelids. Can. J. Zool.
80: 1485-1489.
• Sutton, R.A. and P.D.N. Hebert. 2002. Patterns of sequence divergence in daphniid hemoglobin genes. J.
Mol. Evol. 55: 375-385.
• Adamowicz, S.J., T.R. Gregory, M.C. Marinone and P.D.N. Hebert. 2002. New insights into the distribution
of polyploid Daphnia : the Holarctic revisited and Argentina explored. Mol. Ecol.: 11: 1209-1217.
• Hardie, D.C., T.R. Gregory and P.D.N. Hebert. 2002. From pixels to picograms: a beginner’s guide to genome
quantification by Feulgen image analysis densitometry. J. Histochem. and Cytochem. 50: 735-749.
• Hebert, P.D.N., E.A. Remigio, J.K. Colbourne, D.J. Taylor and C.C. Wilson. 2002. Accelerated molecular
evolution in halophilic crustaceans. Evolution 56: 909-926.
• Cristescu, M.E.A. and P.D.N. Hebert. 2002. Phylogeny and adaptive radiation in the Onychopoda
(Crustacea: Cladocera): evidence from multiple gene sequences. J. Evol. Biol. 15: 838-849.
• Cywinska, A. and P.D.N. Hebert. 2002. Origins of clonal diversity in the hypervariable asexual ostracod
Cypridopsis vidua. J. Evol. Biol. 15: 134-145.
• Hebert, P.D.N. and M.E.A. Cristescu. 2002. Genetic perspectives on invasions: the case of the Cladocera.
Can. J. Fish. Aquat. Sci. 59: 1229-1234.
• Remigio, E.A., D.A.W. Lepitzki, J.S. Lee and P.D.N. Hebert. 2001. Molecular systematic relationships and
evidence for a recent origin of the thermal spring endemic snails Physella johnsoni and Physella wrighti
(Pulmorata: Physidae). Can. J. Zool. 79: 1941-1950.
• Remigio, E.A., P.D.N. Hebert and A. Savage. 2001. Phylogenetic relationships and remarkable radiation in
Parartemia (Crustacea: Anostraca), the endemic brine shrimp of Australia: evidence from mitochondrial
DNA sequences. Biol. J. Linn. Soc. 74: 59-71.

Save NatureConserve the ecosystem