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DNA Barcoding
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.
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DNA Barcoding
- 1. DNA Barcoding Karan VeerSingh 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 appropriatetarget 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 inmany forms Identifiable adults Juvenile stages Processed products
- 5. An Internal IDSystem 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 Editingand 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 leads 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 reactionMixture & 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 1min72°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 Guidefor 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 atNBFGRFISH-BOL Team at NBFGR
- 21. First International Trainingon “DNA Barcoding ofFirst International Training on “DNA Barcoding of Marine Life” organized at NBFGR, Lucknow, IndiaMarine Life” organized at NBFGR, Lucknow, India
- 23.