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dna-diagnostics

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How to use computational tools for diagnostics in broader perspective

How to use computational tools for diagnostics in broader perspective

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  • 1. Bioinformatic approach for DNA signature based diagnosticsDinesh KumarB.Sc. Hons Zoo(BHU), M.Sc. Biotechnology(BHU), Ph.D. Biotechnology (BHU), PDF(USA), PGDIPR, PGDTMASenior Scientist(Biotechnology)Centre for Agricultural Bioinformatics (CABin), IASRI, Pusa, New Delhi-110 012
  • 2. Focus of concept Recognise limitations of “parasitological” diagnosis. Recognise new molecular methods developed. Identify 3 main methods – biochemical, antibody & DNA – based.
  • 3. Why identify parasites? Treatment. Epidemiology. Control measures. Fundamental research.
  • 4. Few popular examples of individual DNAdiagnostics * Rajiv Gandhi Assassination Case (Chennai, Tamil Nadu), * Naina Sahni or the Tandoor case (New Delhi * Priyadarshini Mattoo (New Delhi), * Sishu Vihar Child adoption case (Hyderabad, Andhra Pradesh), * Black Buck killing case (Jodhpur, Rajasthan) * Beanth Singh Assassination Case (Punjab) * ND Tiwari – Biological father hood case Aug 7, 2012
  • 5. Some definitions Diagnostic test: sensitive & specific. – Mathematically defined. Sensitivity: capacity of test to make correct diagnosis in known cases. Specificity: capacity to correctly diagnose uninfected individuals.
  • 6. A diagnosis problem?Traditionally diagnosis infectionbased on finding parasite.Problems: Some parasites morphologically indistinguishable. Parasites hidden in host tissue. Low sensitivity.
  • 7. Traditional diagnosis ofMalaria
  • 8. The solution?Current parasitological diagnostic techniques not satisfactory. Need trained staff, equipment, slow throughput. But “gold standard”. Rapid molecular tests being developed.
  • 9. Three types of “molecular” tests.1. Biochemical (first generation).2. Immunological (antibodies).3. Nucleic acid.
  • 10. 1. Biochemical molecular tests: Enzyme patterns.Isoenzymes. Perform same functions BUT different movement on gels. Genetically controlled∴ parasites with different gel patterns genetically distinct. Isoenzymes) are enzymes that differ in amino acid sequence but catalyze the same chemical reaction
  • 11. Isoenzyme Analysis ofChagas’ Disease
  • 12. Assessment of enzyme pattern based diagnosisAdvantages: Simple technique. Large number of typing enzymes available. Many samples typed at same time. Power to distinguish morphologically similar parasites.
  • 13. Enzyme pattern based diagnosis.Disadvantages:Significant tissue needed for analysis → visceral leishmaniasis requires spleen, liver.Technique not rapid → can take days.Sometimes incorrect diagnosis → enzyme labile.Technique simple but equipment expensive.
  • 14. Iso-enzymes separated bycharge: Isoelectric focusingequipment.
  • 15. Enzymes separated by size:SDS-PAGE.
  • 16.  SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique used in biochemistry, genetics and molecular biology to separate proteins according to their electrophoretic mobility
  • 17. 2. Antibody based diagnosis.Rely on identification of specific antibodies.Advantages: Rapid easy field-based tests. Both individual & mass population screening. Ig subclasses – to improve specificity / sensitivity.
  • 18. Antibody based diagnosis.Disadvantages:Cannot distinguish past / present infections.Cannot distinguish morphologically similar parasites.Expensive to develop – significant research prior to commercialization.
  • 19. Classical ab test: Enzyme-LinkedImmunosorbant Assay (ELISA) fordiagnosis. Positive Negative
  • 20. Basic principles of ELISA. Secondary antibody with label Primary antibody BlockAntigen Ag Ag Ag unbound Microtitre plate well sites
  • 21. Example of Antibody based moleculardiagnosis. African Sleeping SicknessAnti-trypanosomal IgM detected by simple / rapidCATT (Card Agglutination Test for Trypanosomiasis) Drop of blood ↓ Mixed with fixed parasites on plastic card ↓ Blue granular deposits = infection
  • 22. CATT Test for African sleepingsickness.
  • 23. Future for Ab-based diagnosis? Non-invasive sampling? – saliva, excreta? Good results for saliva abs for African trypanosomiasis. But will need new tests. Saliva abs not detected in CATT or Latex.
  • 24. 3. DNA based molecular diagnosis. DNA probes. Polymerase Chain Reaction (PCR).
  • 25. PCR in parasite diagnosis.Amplifies target sequences & increases sensitivity.1. Ribosomal DNA/RNA.  Highly sensitive.  No good for closely related species.2. Specific sequences of genomic DNA.  Highly specific for single species - not sensitive.3. Random primer amplification (RADP) PCR.  Very highly sensitive - not specific.
  • 26. Nucleic acid based molecular diagnosis.Advantages: Genomic DNA constant -parasite & hosts unique DNA sequences . Very sensitive - small biopsy. Probes can be designed with flexibility: → Specific - detect single parasite species. → Less specific - detect group of parasites.
  • 27. Nucleic acid based diagnosis.Disadvantages: Expensive - especially PCR . Radioactivity needed: newer non-radioactive probes. PCR can fail: - Contamination & false positives. DNA probes do not distinguish between dead & living parasites
  • 28. Examples of veterinary parasite nucleic acid diagnosis Cryptosporidium parvum protozoa.1. Faecal microscope analysis: limit 50,000 oocysts per g faeces.2. PCR: amplifies ~400 base pair sequence from faeces - sensitivity to 6 oocysts per g faeces.
  • 29. DNA barcoding of animal species diagnostics cytochrome c oxidase subunit I gene (COI) potential barcode. Mitochondrial DNA has signatureAug 7, 2012
  • 30. Identifying flowering plants by species DNA bar code  Kress et al. (2005) -COI sequence- not appropriate for most species of plants slower rate of cytochrome c oxidase I gene evolution in higher plants than in animals”.  Chloroplast has DNA signature of plant species .  nuclear internal transcribed spacer region and the plastid trnH-psbA intergenic spacer as a potential DNA barcode for flowering plants. Some reports supports MatK  DNA barcoding, no master key may be a master keyring, with different kingdoms of life requiring different keys. Aug 7, 2012
  • 31. Microbial species signature Ribotyping -rRNA database-most used eg 16srDNA Designing of primer for gene sequencing. Eg HKG,Topoiso II, Designing of probes for identifications-Eg.real time, microarray In silico development of RFLP test for close species differentiation(our experiences) Aug 7, 2012
  • 32. rRNA genes - the ideal markers for microbial identification  Small subunit - highest order differences (domains)  Large subunit - medium order differences  ITS - low order differences (species/strains?) Small Sub-Unit rRNA (16S)  ubiquitous  1.6 - 2.0kb  good molecular chronometer.  some areas conserved (for priming/alignment)  some areas variable (for resolving differences) Aug 7, 2012
  • 33. Plant pathogen signature•Naked Eye: Colony - shape, colour.•Microscopy: Cell - shape, colour, size•Biochemistry:•Molecular Level: Role of bioinformatics Aug 7, 2012
  • 34. Microsatellite DNA marker in Fungal diagnostics Aug 7, 2012
  • 35. How fungal microsatellite data gives signature Aug 7, 2012
  • 36. Why we need molecular & bioinformatics tool?  Case study of Ug99-signature searchStem rust never sleeps- Norman E. Borlaug , 26th April, 2008, New York Times Aug 7, 2012
  • 37. Can we have DNA based signatures of Ug99?  National Debate!  Global meet at Delhi, Oct, 2008  Action plan? Aug 7, 2012
  • 38. A case study updates-How to identify Ug99?  Puccinia graminis tritici Ug99  DNA signature is the only answer !  DNA signature of Fungi – Private alleles of STR(rare) – STR allele frequency signatures – SNP based signatures(???)  Where is the signature of Ug99 ?? Aug 7, 2012
  • 39. What are the available DNAmarkers to identify Ug99 SSR AFLP Mol Plant Path Latest Nov 2008 issue Aug 7, 2012
  • 40. SSR data- no DNA signature Mol Plant Path Nov 2008 issue Aug 7, 2012
  • 41. AFLP adapters used to generate signature data Mol Plant Path Nov 2008 issue Aug 7, 2012
  • 42. AFLP signatures Mol Plant Path Nov 2008 issueAug 7, 2012
  • 43. No clear signature of Ug55 & 99 by SSR Mol Plant Path Nov 2008 issue Aug 7, 2012
  • 44. AFLP again poor signature b/w Ug99/55 Mol Plant Path Nov 2008 issue Aug 7, 2012
  • 45. DNA signature of Ug99 by combining dataof SSR+AFLP Mol Plant Path Nov 2008 issue Aug 7, 2012
  • 46. Clear DNA signature by Minimum-spanning network analysis Mol Plant Path Nov 2008 issueAug 7, 2012
  • 47. What bioinformatics can do more inUg99 identification? Allele mining of Puccinia graminis tritici Ug99 – USDA is targeting 400 SNP STR mining from Puccinia graminis genome data base STR based signature searchAug 7, 2012
  • 48. Our experiences-DNA based genderdiagnostics using bioinformatics tools Aug 7, 2012
  • 49. Our experiences-DNA based microbial species signature using bioinformaticsHouse Keeping Genes -CLUSTAL-W-signature with spelling mistake- CLEAVER KspAI Bsh1236I Mun I L P P R RR P P R RR P P R RR KEU1 5-AAY ATG ATI ACI GGI GCI GCI CAR ATG GA-3 KEU 2 5-AYR TTI TCI CCI GGC ATI ACC AT-3. KspAI L.paracasei 542,158bp; L.rhamnosa 701bp Bsh1236I L. paracasei 547,153bp; L. rhamnosa 701bp Mun I L. paracasei 594,106bp; L. rhamnosa 701bp New PCR-RFLP test developed for Lactobacillus species differentiation Aug 7, 2012
  • 50. DNA based signature of domestic species Mitochindrial DNA markers used especially for meat identification, poaching of wild animals, adulteration of dairy milk, dairy products(like cheese) of various domestic animal species.Aug 7, 2012
  • 51. STR & breed-specific signatures/profiles Pig-In UK, Signer et al. (2000) - minisatellite probe pCMS12 -three breeds of pig viz Chinese, Meishan, Large White and other European breeds. The linear discrimination analysis revealed that the DNA profiles were breed specific. Fish-In Finland, Primmer et al. (2000) - disputed fish to a specific population out of 4 suspected fish populations using 7 microsatellite loci by Bayesian method with confidence limit of 99%. Sheep-In Spain, Arranz et al. (2001) - Bayesian method with 99.63% accuracy among five Spanish sheep breed viz. Churra, Latxa, Castellana, Rasa-Aragonesa and Merino using 18 microsatellite markers. Horse-In Norway, Bjornstad et al. (2001) -26 microsatellite loci in six breeds of horses, Fjord, Nordland/ Lyngen, Dole, Trotter, Icelandic horse and Shetland pony with more than 95% confidence limit. Cattle-In European countries, Canon et al. (2001) -confidence limit of 99% for 18 local breeds of cattle of different countries; Alistana, Astruriana, Asturiana Valles, Sayaguesa, Tudanca, Avilena Negra-Iberica, Bruna del Pirineus, Morucha, Pirenaica, Retinta of Spain; Alentejana, Barrosa, Maronesa, Mertolenga, Mirandesa of Portugal and Aubrac, Gasconne, Salers of France Camel- In Kenya, Mburu et al. (2003) - 4 breeds using 14 microsatellite loci of camel viz. Somali, Turkana, Rendille, Gabbra) using maximum likelihood method up to 48 % confidence limit.-weak genetic differentiation and gene flow between populations. Dog-In2012 Aug 7, Finland, Koskinen (2003) has assigned breeds of domestic dog using
  • 52. Few examples: Signature applications 1. Application of polymerase chain reaction to detect adulteration of sheeps milk with goats: J Dairy Sci (2005) 88: 3115-20. 2. A novel approach to the quantification of bovine milk in ovine cheeses using a duplex PCR: J Agric Food Chem (2004) 52: 4943-7. 3. Rapid detection of cows milk in sheeps and goats milk by a species- specific polymerase: J Dairy Sci (2004) 87: 2839-45. 4. Identification of cows milk in "buffalo" cheese by duplex polymerase chain reaction. J Food Prot (2002) 65: 362-6. 5. Forensic identification of ungulate species using restriction digests of PCR-amplified mit J Forensic Sci (1995) 40: 943-51. (15 species) 6. Detection of cows milk in goats cheeses inferred from mitochondrial DNA polymorphism Journal of Dairy Research (2001), 68:229-235 7.Application of polymerase chain reaction for detection of goats milk adulteration by milk of cow. Journal of Dairy Research (2001), 68:333-336 Aug 7, 2012
  • 53. DNA based signature of plant variety, example- Basmati rice Basmati rice -aroma compound -2-acetyl-1- pyrroline. Fraudulent traders to adulterate traditional basmati. PCR-based assay similar to DNA fingerprinting in humans allows for the detection of adulterated and non-basmati strains. Its detection limit for adulteration is from 1% upwards with an error rate of ±1.5%. Exporters of basmati rice use purity certificates based on DNA tests for their basmati rice consignments. It was developed by CDFD, Labindia, Worlds First Single-tube, Multiplex(co-amplify eight microsatellite loci) Microsatellite Assay-based Kit for Basmati Authentication. Aug 7, 2012
  • 54. Few examples of individual DNA diagnostics * Rajiv Gandhi Assassination Case (Chennai, Tamil Nadu), * Naina Sahni or the Tandoor case (New Delhi * Priyadarshini Mattoo (New Delhi), * Sishu Vihar Child adoption case (Hyderabad, Andhra Pradesh), * Black Buck killing case (Jodhpur, Rajasthan) * Beanth Singh Assassination Case (Punjab) Aug 7, 2012
  • 55. Acknowledgements• Our team : Prem, Vijay, Nishant, Dhiraj, Vishal, Bhola, Vipin, Udai, Pooja Thank you dineshkumarbhu@gmail.com,

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