Dna barcoding

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
and this presentation gives much introducing about DNA barcodes developed for Prokaryotes and Eukaryotes.
Various barcoding genes which are evolutionary conserved.
techniques to develop a DNA bar-code and its future perspectives
Current technologies and future technologies of DNA barcoding. Applications regarding environment awareness. it also contains 2-3 case studies

1. DNA B a r c odi ng Kandhan . S, M. Tech (Biotechnology) PSG College of Techn ology

2. 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

3. 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

4. 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

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

6. http:www.barcoding.Si.edu

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

8. 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.

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

10. WHERE I’M Nucleus

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

12. 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

13. 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

14. 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 .

15. % 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

16. 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

17. Comparison of Plant Barcode region

18. 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 ,2005 Conrad L. schock at al , 2012

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

20. 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/)

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

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

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

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

25. Sequence Align UPLOAD IN BOLD AND OTHER DATABASE CONVERT TO BARCODE

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

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

28. Hollingworth,2008

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

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

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

32. 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

33. 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

34. 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

35. Case studies

36. Hebert et al,2007

37. R.Sriama and Uma Shaanker ,

38. Bha

39. Case studies

40. CONSERVE OUR ECOSYSTEM This is where we stand today!

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

42. 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 .

43. 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.

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