DNA barcoding uses a short, standardized gene sequence from a uniform region of mitochondrial or nuclear DNA to identify species. It has potential to identify the estimated 10 million eukaryotic species on Earth. The cytochrome c oxidase I (COI) gene region is commonly used for animals. DNA barcoding can identify specimens at all life stages, resolve taxonomic ambiguities, and enable development of electronic field guides. It involves tissue sampling, DNA extraction and amplification, sequencing, and comparing sequences to reference databases. Costs and time for the process are decreasing with new technologies. Global efforts aim to compile barcodes for all known eukaryotes.

1. DNA Barcoding
Karan Veer Singh
NBFGR, Lucknow

2. DNA BarcodingDNA Barcoding
 DNA sequence analysis of a uniform target gene like
the mitochondrial cytochrome oxidase subunit I (COI)
to enable species identification has been referred to as
“DNA Barcoding”, by analogy with the Universal
Product Code (UPC) system barcodes used to identify
manufactured goods.
 DNA barcoding has the potential to be a practical
method for identification of the estimated 10 million
species of eukaryotic life on earth.
 Expected to work at all stages of life, resolve
taxonomic ambiguities, unmasks look-alikes and opens
the way for an electronic handheld field guide, ‘the life
barcoder’.

3. • An appropriate target gene like COI is conserved enough to
be amplified with broad range of primers and divergent
enough to allow discrimination of closely allied species and
it shows comparatively less sequence divergence within a
species compared to the fast evolving genes of mtDNA.
• Alternate target genes are needed for cnidarians ( Hard corals
and some jelly fishes) and plants as these groups show too
little mitochondrial sequence diversity.
• mtDNA of fungi contains introns that can complicate DNA
amplification. Fungi are usually defined based on
morphology rather than on genetic isolating mechanisms.
Thus, the boundaries between named species may not always
correspond with the boundaries of the gene pool. In addition
fungi display a wide range of reproductive strategies,
including sexual, asexual and parasexual (a form of reproduction
in which recombination of genes from different individuals occurs without
meiosis and fertilization) reproduction. Furthermore, hyphal
anastomoses between different strains and morphospecies
make the application of a conventional species definition
especially difficult.
• Global effort, recently initiated to compile DNA barcodes of
all known eukaryotes along with collection of voucher
specimens with authoritative taxonomic identification.

4. Biological specimens
come in many forms
Identifiable
adults
Juvenile stages
Processed products

5. An Internal ID System for All Animals
Typical Animal Cell
Mitochondrion
DNA
mtDNA
Cytochrome b
The Mitochondrial Genome
D-Loop
H-strand
COIII
L-strand
ND6
ND2
COII
Small ribosomal RNA
COI
Cytb
Target
Region
DNA Barcoding – A target region

6. • DNA “Barcode”
• 4 states
• 655 positions
• Universal Product Code
• 10 state
• 11 positions
DNA-based identification system:
DNA Barcoding – The idea

7. DNA Barcoding – The idea
DNA-based identification system:

9.
Fresh/Frozen Time
Tissue Sampling $0.41 10
DNA Extraction $0.34 10
PCR Amplification $0.24 20
PCR Product Check $0.35 5
Cycle Sequencing $1.04 30
Sequencing Cleanup $0.32 5
Sequence $0.40 35
Total: $3.10 115 min
Costs and Time
Towards optimization

10. Sequence COI
Tissue Sample
Editing and Aligning COI sequence and
barcoding
Fig 1. Various steps of DNA Barcoding
PCR COI
Extract DNA

11. Folmer et al., 1994
5'- TAAACTTCAGGGTGACCAAAAAATCA - 3'HCO2198
5'- GGTCAACAAATCATAAAGATATTGG - 3'LCO1490
Sequence (5’ – 3’)Primer Name
Folmer et al., 1994
5'- TAAACTTCAGGGTGACCAAAAAATCA - 3'HCO2198
5'- GGTCAACAAATCATAAAGATATTGG - 3'LCO1490
Sequence (5’ – 3’)Primer Name
Palumbi et al., 1991
L 5’ – CCTGCAGGAGGAGGAGAYCC – 3’COIf
H 5’ – AGTATAAGCGTCTGGGTAGTC – 3’COIa Palumbi et al., 1991
L 5’ – CCTGCAGGAGGAGGAGAYCC – 3’COIf
H 5’ – AGTATAAGCGTCTGGGTAGTC – 3’COIa
Primers for COI
Ward et al., 2005
ACTTCAGGGTGACCGAAGAATCAGAAFishR2
TAGACTTCTGGGTGGCCAAAGAATCAFishR1
TCGACTAATCATAAAGATATCGGCACFishF2
TCAACCAACCACAAAGACATTGGCACFishF1
Ward et al., 2005
ACTTCAGGGTGACCGAAGAATCAGAAFishR2
TAGACTTCTGGGTGGCCAAAGAATCAFishR1
TCGACTAATCATAAAGATATCGGCACFishF2
TCAACCAACCACAAAGACATTGGCACFishF1

12. 55.988 X
75%
= 41.99 nmoles
↓
This multiplied by 5
=41.991 X 5 = 209.955 (add this much amount of
TE/H20(autoclaved))
(To prepare the stock)
Desalting lead s to loss of 25% oligos hence
actual (available) concentration
Primer (desalted dilution)Primer (desalted dilution)
(give order for 50nm)(give order for 50nm)
On the vial 0.27994nm/µL if reconstituted in
200µL of TE/ H20
↓
0.27994 X 200 = 55.988
Working
Solution
= 10µL od FP stock
10µL of RP stock
180µL of autoclaved H20
-------
Total 200µL (concentration 5 p moles each
primer / 25µL PCR mix)

13. mtDNA PCR reaction Mixture & ConcentrationmtDNA PCR reaction Mixture & Concentration
Volume perVolume per
reactionreaction
Double distilled waterDouble distilled water 18.018.0µµLL
AssayAssay buffer (10X; Genei, Bangalore, India)buffer (10X; Genei, Bangalore, India)
(100mM Tris, 500mM KCl, 0.1% gelatin, pH9) (final(100mM Tris, 500mM KCl, 0.1% gelatin, pH9) (final
conc. 1X)conc. 1X)
2.52.5µµLL
dNTPs (Genei, Bangalore, India) (200 mM)dNTPs (Genei, Bangalore, India) (200 mM) 2.02.0µµLL
Primer (forward & reverse) (Operon Technologies,Primer (forward & reverse) (Operon Technologies,
USA) (~ 10.0 pmoles/USA) (~ 10.0 pmoles/µµl)l)
0.50.5µµLL
MgClMgCl22 (1.5mM )(1.5mM ) 0.50.5 µµll
TaqTaq polymerase (Genei, Bangalore, India) (3Units/polymerase (Genei, Bangalore, India) (3Units/
µµll))
0.50.5µµLL
Template DNA (25ng)Template DNA (25ng) 1.01.0µµLL
Total volumeTotal volume 25.025.0µµLL

14. --
--4°CSoak
1
10 min72°CElongated extension
1 min72°CExtension
30 sec54° CAnnealing
35cycles
30 sec94°CDenaturation
1
2 min95°CInitial step denaturation
CyclesTimeTemperatureSteps
--
--4°CSoak
1
10 min72°CElongated extension
1 min72°CExtension
30 sec54° CAnnealing
35cycles
30 sec94°CDenaturation
1
2 min95°CInitial step denaturation
CyclesTimeTemperatureSteps
PCR Steps for COI amplification

15. Primer Name Sequence (5’ – 3’)
18SrRNA forward AACCTGGTTGATCCTGCCAGT
18SrRNA reverse TGATCCTTCTGCAGGTTCACCTAC
Barcoding Electives – other genes

16. Producing Barcode Data: 2006Producing Barcode Data: 2006
ABI 3100 capillary
automated sequencer
• Hundreds of samples per day
• costing several dollars per sample

17. Producing Barcode Data: 2008Producing Barcode Data: 2008
Faster, more portable: Hundreds of samples per hour
Integrated DNA microchips Table-top microfluidic systems

18. Producing Barcode Data: 2010?
Hand-held BarcoderHand-held Barcoder
Barcode data anywhere, instantly
 Data in seconds to
minutes
 Pennies per sample
 Link to reference
database
 A taxonomic GPS
 Usable by non-
specialists

19. A Field Guide for the Third Millennium
Consortium site:
www.cbol.org
Rock U site with Barcode Blog
http://phe.rockefeller.edu/
barcod/
Guelph site:
www.barcodinglife.org

20. FISH-BOL Team at NBFGRFISH-BOL Team at NBFGR

21. First International Training on “DNA Barcoding ofFirst International Training on “DNA Barcoding of
Marine Life” organized at NBFGR, Lucknow, IndiaMarine Life” organized at NBFGR, Lucknow, India

23. THANK YOU