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1. University of Gondar Institute of Biotechnology
Department of Biotechnology
Introductions to Omics Course
By:-Tewodros Eshetie(BSc, in Biotechnology, MSc in Medical biotechnology)
[email protected]/[email protected]
1

2. •Chapter 1: Introduction to Omics Science
2

3. What is omics ?
•Thecompletesequencingofhumangenomehasusheredinneweraofsystembiology
referredtoasomicstechnology.
•Theterm“omic”derivedfromthelattinsuffix“ome”meaningmassormany.Thus
OMICSinvolveamass(largenumber)ofmeasurementperendpoint.
•Omicsreferstovariousbranchesofsciencedealingwith“Omes”
3

4. •In biological context, suffix -omics is used to refer a study of large set of
biological molecule
•The broad analysis of large biological molecules was needed to be studied in detail
as the conclusion of human genome project (HGP) in 2001.
•Omesarethecompletecollectionofobjectsorthewholesystemunderstudy
➢Genome
➢Transcriptome
➢Proteome
➢Metabolome
•Omicsreferstothecollectivetechnologiesusedtoexploretheroles,
relationships,andactionsofvarioustypesofmoleculesthatmakeupthecellsof
anorganism
4

5. cont’d…
•OmicsprovidethetoolsneededtolookatthedifferencesinDNA,RNA,proteins
andothercellularmoleculesbetweenspeciesandamongindividualsofaspecies
•Both“omics”and“ome”arerecentlyemergedfromtheiroriginsassimple
suffixestofullyacceptednouns
•Thesuffix“ome”isnowinwidespreaduseandisthoughttohaveoriginatedasa
backformationfromgenome
5

6. cont’d…
•Thetermgenome
✓Istheoldestandbestknownnameoftheomesfamily
✓Wasintroducedin1920bytheGermanbotanistHansWinklerfrom
•Gen(“gene”)+(chromos)om(chromosome)
•The first genome was completely sequenced by sanger in Cambridge, UK, in the
1970s.
•Genome is the most fundamental part of many omics and it is defined as an
organism's genetic material.
6

7. Cont’d…
•Generallyomicsistheemergingtechnologyforunderstandingthe
behaviourofcells,tissues,organsandthewholeorganismatthe
molecularlevelusingmethodssuchasgenomicsproteomics…
7

8. 2.Types of omics studies
•Therearefourmajortypesoftoolsthatarecommonlyperformedordoneinomics
technology;
➢Genomics
➢Transcriptomics
➢Proteomics
➢Metabolomics
8

9. 2.1.Genomics
•Genomicsisthestudyofanorganism’sgenome,orgeneticmaterial.
•Genomecanbedefinedasthecompletesetofgenesinsideacell,andintroduced
byHana'sWinkler1920.
•Thegenomecontainsthecodedinstructionsnecessaryfortheorganismtobuild
andmaintainitself.
•Genomicsemergedasanewfieldofmolecularbiologywherenoveltechnologies
wereexploitedinordertounderstandthecomplex,biologicalfunctionofthe
genome.
•Itrevealedthestaticsequencesofgenes.
9

10. 2.2.transcriptomics
•ThetranscriptomeisthesetofallRNAmolecules,includingmRNA,
rRNA,tRNA,andothernon-codingRNAproducedinoneora
populationofcells.
•Studyoftranscriptomeiscalledtranscriptomics.
•Techniquesinclude;
➢Expressionprofiling
➢DNAmicroarrays
10

11. 2.3.Proteomics
•Proteomicsisthelarge-scalestudyofproteins,particularlytheirstructuresand
functions.
•Theproteomeistheentirecomplementofproteinsexpressedbyacellortissue
type.
•Proteomicsconfirmsthepresenceoftheproteinandprovidesadirectmeasureof
thequantitypresent.Itfocusesontheidentification,localization,andfunctional
analysisoftheproteinmake-upofthecell.
•Itseekstoprofileeveryproteinbeingexpressedinacellortissue.
11

12. 2.4.Metabolomics
•Metabolomicsisthesystematicstudyoftheuniquechemicalfingerprintsthat
specificcellularprocessesleavebehind,thestudyoftheirsmall-molecule
metaboliteprofiles.
•Themetabolomerepresentsthecollectionofallmetabolitesinabiologicalcell,
tissue,organororganism,whicharetheendproductsofcellularprocesses.
•Metabolomereferstothecompletesetofsmall-moleculemetabolitessuchas
metabolicintermediates,hormonesandothersignalingmolecules,andsecondary
metabolitestobefoundwithinabiologicalsample.
12

13. Basic Principle of Omics
13

14. Cont’d…
14

15. Cont’d…
15

16. Cont’d…
16

17. Cont’d…
17

18. Thank you
18

19. •Chapter two: Genomics
19

20. ❑Objectives:
❖Aftercompletionofthissessionthestudentwillbeableto;
➢DefineanddescribeGenomics
➢Explaingenemappingandsequencing
➢Describedifferenttypesofgenomics
20

21. 1.Introduction
•Thedevelopmentofmoleculartechniquestoanalysegenesandexpression
revolutionizedexperimentalbiology.
•OnceDNAsequencingtechniquesweredevelopedscientistrealizedthepossibility
todeterminingthesequencesofwholegenomes.
•Theinformationinthegenesofanorganism(genotype)isresponsibleforthefinal
physicalmakeup(phenotype)oftheorganism.
•SodecodingtheDNAisonesteptowardsunderstandingtheprocess
•Howeverbyitselfitdoesnotspecifyeverything.
21

22. What is genomics?
•Thebranchofbiologicalsciencesthatdealswiththestructureand
informationencodedinthegenomesoforganism.
•Itsnewandexcitingsciencethatdealswiththediscoveryandnoting
ofallthesequencesintheentiregenomeofaparticularorganism
•Genomicsreallycameintoitsownwiththecompletionof
-humangenomeprojectwiththeaimtosupplyprecisemapping
andthecompletesequenceofhumangenome.
22

23. Cont’d…
•The topics of genomics range from genome mapping, sequencing and
functional genomic analysis to comparative genomic analysis.
•Itisthefirstoftheomicsbranchestohavebeendefinedintially
mainlyinvolvedinanalysingDNAsequencingprojectsdataaimedto
mapandsequencethesetofentiregenomes
•Thestudyofthestructure,functionandexpressionofallgenesinan
organism.
•genomicsisthereforethestudyofthegeneticmakeupoforganisms
23

24. Cont’d…
•Genomicsisanentrypointforlookingattheotheromicssciences.
•Thegenomesoforganismsarethelongestunitsofdatacarrying
information.
•AlthoughRNAandproteinpolymersseemtoexhibitamuchmore
variablefunctionalandspatialstructurethanDNAtheirlinearcontent
isalwaysacopyofashortfragmentofthegenome.
24

25. Cont’d…
•ThegenomeisalltheDNAinacell
•AlltheDNAontheallthechromosomes
•Includesgenesintergenicsequencesrepeats
•SpecificallyitisalltheDNAinanorganelle
•Eukaryotecanhave2-3genomes
•Nucleargenome
•Mitochondrialgenome
•Plastidgenome
•Ifnotspecifiedgenomeusuallyreferstothenucleargenome
25

26. •Why should we study genome?
•What is the difference between genome and transcriptome
proteome and metabolome?
•What makes genome different from the others?
26

27. Why should we study genome?
➢Eachandeveryoneisauniquecreation.howsimilarwearehow
differentweare
➢Todeterminethefunctionofgene
➢TodetermineStructureofgene
➢Tomakecomparision
➢Life’slittlebookofinstructionstodeterminewerewearefrom
27

28. Cont’d…
•The experimental techniques and tools to study the genome structure
and function include
•Gene mapping
•Sequence technique
•DNA microarrays
•PCR
•Probe
•Hybridization e.t.c
28

29. Cont’d…
❖Experimentaltechniquesmustbecombinedwithinformaticandmathematical
modellingtoolsinorderto;
•Organizeandstoretheresults
•constructandmanagegenomicdatabases
•Searchforstructureandorderandperformcomparisonsand
•Comeupwithnewhypothesisbasedonthedata
29

30. 1.1.Gene mapping
•Genemappingreferstomappingofthegenestospecificlocationonchromosome.
•Itsveryimportantintheunderstandingofgeneticdisease
•Suchmapindicatethepositionofgenesinthegenomeandalsodistancebetween
them.
•Therearetwotypesofgenemapping;
➢geneticmapping–usinglinkageanalysistodeterminetherelativeposition
betweentwogenesonchromosome
30

31. Cont’d…
➢Physicalmapping-usingallavailabletechniquesorinformationtodeterminethe
absolutepositionofgeneonchromosome.
•Genetic distance is measured by frequency of crossing over between loci on the
same chromosome
•One map unit= one centimorgan(CM)=1% recombination between them
31

32. ❑Uses of Gene Mapping
•Identify genes responsible for diseases.
✓Heritable diseases
✓Cancer
•Identify genes responsible for traits.
✓Plants or Animals
✓Disease resistance
✓Meat or Milk Production
32

33. 1.2.DNA sequencing
❖DNAsequencing:Processofdeterminingthepreciseorderofnucleotideswithina
DNAmolecule.
❖Itincludesanymethodortechnologythatisusedtodeterminetheorderofthefour
bases
Adenine(A)
Guanine(G)
Cytosine(C)
Thymine(T)
❖AdventofrapidDNAsequencingmethodshasgreatlyacceleratedbiologicaland
medicalresearchanddiscovery
33

34. Cont’d…
❖KnowledgeofDNAsequenceshasbecomeindispensableforbasicbiologicalresearch,
andinnumerousappliedfields.
✓Diagnostics
✓Biotechnology
✓Forensicbiology
✓Biologicalsystematics.
❖TherapidspeedofsequencingattainedwithmodernDNAsequencingtechnologyhas
beeninstrumentalinthesequencingofcompleteDNAsequences,orgenomesof
numeroustypesandspeciesoflife,
-includingthehumangenomeandothercompleteDNAsequencesofmany
animal,plant,andmicrobialspecies.
34

35. Cont’d…
❖History;
➢Several notable advancements in DNA sequencing were made during the 1970s.
➢Frederick Sanger developed rapid DNA sequencing methods at Cambridge, UK
and published a method for "DNA sequencing with chain terminating inhibitors" in
1977.
➢Walter Gilbert and Allan Maxam at Harvard also developed sequencing
methods, including one for "DNA sequencing by chemical degradation"
➢The first full DNA genome to be sequenced was that of bacteriophage φX174 in
1977.
35

36. Cont’d…
➢MedicalResearchCouncilscientistsdecipheredthecompleteDNAsequenceof
theEpstein-Barrvirusin1984,findingittobe170thousandbase-pairslong.
➢LeroyE.Hood'slaboratoryattheCaliforniaInstituteofTechnologyandSmith
announcedthefirstsemi-automatedDNAsequencingmachinein1986.
➢FollowedbyAppliedBiosystems'marketingofthefirstfullyautomated
sequencingmachine,theABI370,in1987.
➢By1990,theU.S.NIHhadbegunlarge-scalesequencingtrialsonMycoplasma
capricolum,Escherichiacoli,Caenorhabditiselegans,andSaccharomyces
cerevisiaeatacostofUS$0.75perbase.
36

37. Cont’d…
➢Several new methods for DNA sequencing were developed in the mid to late 1990s.
➢These techniques comprise the first of the "next-generation" sequencing methods.
➢In 1996, PålNyrénand his student Mostafa Ronaghi at the Royal Institute of Technology
in Stockholm published their method of pyrosequencing.
➢In 2000 Lynx Therapeutics published and marketed "Massively parallel signature
sequencing", or MPSS.
➢This method incorporated a parallelized, adapter/ligation mediated, bead-based sequencing
technology and served as the first commercially available "next-generation“ sequencing
method.
37

38. Why we need to sequence genome?
❑Whysequenceagenome?
•Tohavecompletegeneticblueprintoftheorganism
•Becausethesequenceofnucleotidesinthegenomeareimportant
informationtodetermine;
-Howdifferentregionsofthegenomecontrolthedevelopmentand
functionofanorganism.
•Toidentifydifferenceamongindividual
38

39. ❑Use of sequencing
➢DNA sequencing used to determine the sequence of individual genes, larger genetic
regions, full chromosomes or entire genomes.
➢provide the order of nucleotides in DNA or RNA isolated from cells of animals,
plants, bacteria or virtually any other source of genetic information.
➢The resulting sequences may be used by researchers in molecular biology or genetics
to further scientific progress or may be used by medical personnel to make treatment
decisions or aid in genetic counseling.
39

40. 2.Types of genomics
➢Genomediffersignificantlybetweenspeciesinsizetopologytypes
andfunction.
❖Genomicsisclassifiedas;
▪Functionalgenomics
▪Comparativegenomics
▪Structuralgenomics
40

41. 2.1.Functional genomics
•Functionalgenomicsislargelyexperimentbasedgenomestudywithafocuson
gene
•Functionalgenomicsisthestudyofthefunctionsofgeneatthegenomelevel
usinghighthroughputapproaches.
•“highthroughput,”issimultaneousanalysisofallgenesinagenome
•Thisfeatureisinfactwhatseparatesgenomicsfromtraditionalmolecularbiology,
whichstudiesonlyonegeneatatime.
41

42. Cont’d…
•allowsdeterminationofthefunctionsofgenesthatwerepreviously
uncharacterized.
•Inshort,functionalgenomicsprovidesinsightintothebiologicalfunctionsofthe
wholegenomebyusinghighthroughputexpressionanalysisalsotermedas
transcriptiomeanalysiswhichuses
•Transcriptomeanalysiscanbeconductedbytwoapproaches:
1)sequencebasedapproaches
2)microarraybasedapproaches
42

43. Expressed sequence tag
•Expressedsequencetags:-ESTsareshortsequencesofcDNAtypically200-400
nucleotidesinlength.
•Obtainedfromeither5’endor3’endofcDNAinsertsofcDNAlibrary.
•cDNALibrariescanbepreparedbyreversetranscriptionofmRNAbyusingoligo
(dT)primersthathybridizewiththepoly(A)tailofmRNAsandligationofthecDNAs
tocloningvectors.
•usedtoidentifytheunknowngenexpression,genomemapconstructionand
characterizationofexpressedgene
43

44. microarray-based approaches
•Themostcommonlyusedglobalgeneexpressionprofilingmethodincurrent
genomicsresearchistheDNAmicroarray-basedapproach
•Amicroarray(orgenechip)isaslideattachedwithahigh-densityarrayof
immobilizedDNAoligomers(sometimescDNAs)representingtheentiregenome
ofthespeciesunderstudy
•Each oligomer is spotted on the slide and serves as a probe for binding to a
unique, complementary cDNA.
44

45. Cont’d…
•TheentirecDNApopulation,labeledwithfluorescentdyesorradioisotopes,is
allowedtohybridizewiththeoligoprobesonthechip
•Theamountoffluorescentorradiolabelsateachspotpositionreflectstheamount
ofcorrespondingmRNAinthecell.
•Using this analysis, patterns of global gene expression in a cell can be examined
•Sets of genes involved in the same regulatory or metabolic pathways can
potentially be identified.
45

46. Cont’d…
46

47. 2.2.Comparative genomics
•Comparativegenomicsisthestudyoftherelationshipofgenomestructureandfunction
acrossdifferentbiologicalspeciesorstrains.
•ItStudiesthedifferencesandsimilaritiesb/ngenomeofdifferentorganisms
•Comparativegenomicsincludescomparisonofgenelocationandgenecontentfromd/t
genomes.
•Itisanattempttotakeadvantageoftheinformationprovidedbythesignaturesof
selectiontounderstandthefunctionandevolutionaryprocessesthatactongenomes.
47

48. Cont’d…
❖WhyweneedtoCompare?
•Needtobetterunderstandtheindividualgenomes
•Tounderstandthefunctioningofindividualgenes
•Toderiveacomparativestudyofbasicfunctions
•Tobetterunderstandevolutionaryprocesses
48

49. General genomic comparisons
Organism Genome Size (Bases) Estimated Genes
Human (Homo sapiens) 3 billion 30,000
Laboratory mouse
(M. musculus)
2.6 billion 30,000
Thale cress (A. thaliana) 100 million 25,000
Roundworm (C. elegans) 97 million 19,000
Fruit fly (D. melanogaster) 137 million 13,000
Yeast (S. cerevisiae) 12.1 million 6,000
Bacterium (E. coli) 4.6 million 3,200
Human immunodeficiency virus
(HIV)
9700 9
49

50. 2.3.Structural genomics
➢StructuralGenomics:dealswiththestudyofthestructureofentiregenomeofan
organism.Inotherwords,itdealswiththestudyofthegeneticstructureoftheeach
chromosomeofthegenome.
➢Itcharacterizesthephysicalnatureofwholegenome.
➢Itincludesthegeneticmapping,physicalmappingandsequencingofwholegenomes.
➢Itdescribesthe3Dstructureofeveryproteinencodedbyagivengenome.
➢Theprincipaldifferenceb/nstructuralgenomicsandtraditionalstructuralpredictionisthat;
-structuralgenomicsattemptstodeterminethestructureofeveryproteinencodedby
thegenomeratherthanfocusingononeparticularprotein.
50

51. Cont’d…
•Structuralgenomicstakesadvantageofcompletedgenomesequencesinorderto
determineproteinstructure.
•Thegenesequenceofthetargetproteincanbecomparedwiththesequencehaving
knownstructurefromwhichthestructureofunknownproteinsequenceisinferred
orpredicted.
•Structuralgenomicshasroleindeterminationoffunctionofaprotein,Usedin
drugdiscoveryandinproteinengineeringonalargescale,Interpretationof
proteinstructure.
51

52. Cont’d…
➢Becauseproteinstructureiscloselylinkedwithproteinfunction,thestructural
genomicshasthepotentialtoinformknowledgeofproteinfunction.
➢Inadditiontoelucidatingproteinfunctions,structuralgenomicscanbeusedto
identifynovelproteinfoldsandpotentialtargetsfordrugdiscovery.
➢Ithelpsthescientificcommunitytogetsimmediateaccesstonewstructures,as
wellastoreagentssuchasclonesandprotein.
➢Structuralgenomicscanalsotakemodellingbasedapproachthatrelieson
homologybetweentheunknownproteinandasolvedproteinstructure.
52

53. Genome organization and evolution
53

54. ❑Objectives:
❖Aftercompletionofthissessionthestudentwillbeableto;
➢DefineanddescribeGenomeorganization
➢ListandanalyzeGenomemappingmethods
➢ExplainmarkersforGenomemapping
54

55. 3.Genome organization and evolution
➢Thehereditarymateriali.e.DNA(deoxyribonucleicacid)ofanorganismiscomposedof
asequenceoffournucleotidesinaspecificpattern,whichencodesinformationasa
functionoftheirorder.
➢Thishereditarymaterialgetschangedthroughaprocesscalledevolution.
➢Genomeevolutionisaprocessbywhichagenomechangesinstructure(sequence)orsize
overtimethrough;mutation,genetransfer,reproduction
➢GenomicorganizationreferstothelinearorderofDNAelementsandtheirdivision
intochromosomes.
55

56. Cont’d…
➢"Genomeorganization"canalsorefertothe3Dstructureofchromosomeandthe
positioningofDNAsequenceswithinthenucleus.
➢Chromosomesarethestructuresthatcontainthegeneticmaterial
➢TheyarecomplexesofDNAandproteins
➢Thegenomecomprisesallthegeneticmaterialthatanorganismpossesses.
➢Organismshaveavastarrayofwaysinwhichtheirrespectivegenomesareorganized.
-sogenomeorganizationvariesindifferentorganism.
56

57. Cont’d…
➢Basedontheirbasicchemistry,structure,andhereditarymaterial,allcellsonEarthfall
intotwogroups;EukaryotesandProkaryotes
➢Eukaryotesaremostlymulti-cellularorganismsthataredistinguishedfromprokaryotes
bythepresenceofamembrane-boundorganellesthatperformvariousfunctionsfor
thecell.
➢Nucleusisoneofthoseorganelleswherethenucleargeneticmaterialofeukaryotesis
found.
57

58. Cont’d…
➢Eukaryoticcellsaretypicallymuchlargerthanprokaryoticcells,onaverageabouttentimes
largerandundergosexualreproduction
➢TheDNAinprokaryotesaresmallerinsize,circularandpresentincytoplasm
-Prokaryoticgenomeisverycompact(highlycondensed)andcontainverylittlenon-
codingDNAsequences
➢While,theeukaryoticDNAislargerinsize,linear,morecomplexandlocatedin
membrane-boundednucleus.
-inEukaryoticgenomeshareofnon–codingpartsofDNAisabout98%,in
prokaryotesitisonly12%.
58

59. 3.1.Prokaryotes genome organization
➢Unicellularcell
➢TheDNAinprokaryotesaresmallerinsizeandpresentincytoplasm,comparedwith
eukaryoticgenome
➢Prokaryoticgenomeisverycompact(highlycondensed)andcontainverylittlenon-
codingDNAsequences
➢Containsinglecircularchromosomecalled,haploidorganism
➢DNAisnotassociatedwithhistoneproteinandhasoperon
59

60. Cont’d…
❖Theprokaryotesgenomeorganizationclassifiedinto3stages;
1.Nucleoid;aparticularareainwhichprokaryotesassembletheirgeneticmaterial,lacks
protectivemembraneanditsirregularinshape.
➢ItusuallycontaincontainonlyonechromosomecomposedofDNA,RNAandother
protein
➢ProkaryotesmayalsocontainextrachromosomalDNAmoleculecalledplasmid
➢Theseareindependentlyreplicatingsmaller,circularDNAfragmentsthataredispensable.
60

61. Cont’d….
2.Supercoiling
➢Prokaryoticgenomehavetofitintoaverytinyspace
-thecircularE.colichromosomehascircumferenceof1.6mmwhereasanE.coli
celljusthas0.25microm.
➢Inordertopackageitselfintothissmallerspace,thegenomeissupercoiled
➢SupercoilingisunderwindingofDNAstrand,controlledbytwoenzymes
DNAgyraseandtopoisomeraseI
61

62. Cont’d…
3.Operon
➢AnoperonisregionofDNAthatcontainsmultiplegenesthatarecoregulated.
➢Thisallowsforsimultaneousregulationofthegeneclusterasthegenesshareacommon
promotorthatcanberecognizedbytheRNApolymerase.
62

63. 63

64. 3.2.Eukaryote's genome organization
➢Multicellularcell
➢TheDNAineukaryotesarelargerinsizeandpresentinnucleus,comparedwith
prokaryoticgenome
➢InadditiontoDNAcontainedinthenucleusandribosomes,eukaryoteshaveasmall
mitochondrialandchloroplastDNA
-because eukaryotes have mitochondria (but prokaryotes do not have)
➢Containmultiplelinearchromosomecalled,Diploidorganism
➢DNAisassociatedwithproteinhasnooperon
64

65. Cont’d…
•The Eukaryotes genome organization classified into 4 stages;
1.Nucleosome formation
➢eukaryotic DNA combines with basic protein molecule called histone ,to form a structure
known as nucleosome.
➢Its wrapped structure of 146bp of DNA
➢This structure contain H2A,H2B,H3 and H4 types of histone protein and also…
➢H1 is called linker histone protein
65

66. Cont’d…
66

67. Cont’d…
2.Solenoidfiber
➢Nucleosomefoldedthemselvestoformdense,tightlypackedfiberlikestructurecalled
themsolenoidfiberorhelixmodel
➢Wherenucleosomearrangedinzigzagpattern
➢Thediameterofthisfibreis30nm
67

68. Cont’d…
3.Chromatinfibre
➢ChromatiniscomposedofDNAandprotein,mostlybasicproteins.
➢Afibrechromatinis300nmindiameter,itsstructurealsoknownasreplicatingunitof
nucleosome.
➢ChemicalcompositionofchromatincontainsDNA(20-40%),RNA(5-10%),protein(55-
60%)mostlyhistoneand20%ofnon-histoneprotein
➢Mainlytherearetwotypesofchromatinarethere;EuchromatinandHertochromatin.
68

69. Cont’d…
4.ChromatidandChromosomeformation
➢Thehigherorderofchromatinstructure.
➢itis700nmindiameterandalsocalledchromatid.
➢Chromatidsareconnectedbycentromereandformchromosomewhichis1400nmin
diameter
➢Chromosomecontain3parts;
1.Twoarms(chromatids)
2.Centromeres(regionholdschromatin)
3.Telomers(endofthechromosome)
69

70. Eukaryotes nuclear genome organization
70

71. 3.3.Difference between eukaryotic and prokaryotic genome
Prokaryotic genome Eukaryotic genome
They are small in size They are larger in size
Contain single circular chromosomeContain multiple linear chromosome
They are haploid They are diploid
DNA is not associated histone proteinDNA associated with histone protein
They are condensed in nucleoid of
cytoplasm
They are condensed in membrane bound
nucleus
More than 90% coding sequence Only 3% coding sequence
71

72. 3.4.Genome Mapping
•Genomemappingisdeterminingthelocationofgenesandthedistancesbetweengenesona
chromosome.
•Itisveryimportantintheunderstandingofgeneticdiseases.
➢In1911,byThomasHuntMorgan,geneforeyecolorwaslocatedontheXchromosomeof
fruitfly.
➢E.B.Wilsonattributedthesex-linkedgenesresponsibleforcolor-blindnessandhemophilia
inhumanbeingstobelocatedontheX-chromosome
•Theessenceofallgenomemappingistoplaceacollectionofmolecularmarkersontotheir
respectivepositionsonthegenome.
72

73. 3.4.1.Types of Genome mapping
➢Therearetwotypesofgenomemapping:GeneticandPhysicalmapping
1.Geneticsmapping:lineardescriptionofDNAmarkers/genesonagivenchromosome
withcloselyplacedmarkersbeinginheritedtogethermoreoften.
✓Itillustratetheorderofgenesonachromosomeandtherelativedistancesbetweenthose
genesofmultipletraits,
-suchashaircolorandeyecolor,throughseveralgenerations.
73

74. Cont’d…
•Therationalbehindgeneticmappingisthatthecloserthetwogeneticmarkersare,the
morelikelytheyareinheritedtogetherandarenotseparatedinageneticcrossingevent.
•ThedistancebetweenthetwogeneticmarkersismeasuredincentiMorgans(cM),whichis
thefrequencyofrecombinationofgeneticmarkers
•AlfredSturtevantcreatedthefirstgeneticmapofachromosomefromthefruitfly
(Drosophilamelanogaster)in1913.
•Hedeterminedthatgeneswerearrangedonchromosomesinalinearway,likebeadsona
necklace,andthatgenesforspecifictraitsarelocatedinparticularplaces.
74

75. Cont’d…
•Heproposedthatthefrequencyof‘crossingover’(recombination)betweentwo
genescouldhelpdeterminetheirlocationonachromosome.
•Herealizedthatgenesthatwerefarapartonachromosomearemorelikelytobe
inheritedseparatelysimplybecausethereisalargerregionoverwhich
recombinationcanoccur.
•Inthesameway,genesthatareclosetoeachotheronthechromosomearemore
likelytobeinheritedtogether.
75

76. Cont’d…
76

77. Cont’d…
•Byfindingouthowoftenvariouscharacteristicsareinheritedtogetheritispossibleto
estimatethedistancebetweenthegenes.
•Example:-Iftwo(ormore)characteristicswereseentobefrequentlyinheritedtogetherin
afamily,
-forexamplelight-coloredhairandblueeyes,itsuggestedthatthegenesforthetwo
characteristicswereclosetogetheronaparticularchromosome.
•Amapofwherethegenesareinrelationshiptoeachotheronthechromosomescanthen
bedrawn.Thisiscalledagenelinkagemap.
77

78. Cont’d…
•Genes that are on the same chromosome are said to be ‘linked’ and the
distance between these genes is called a ‘linkage distance’.
•The smaller the distance the more likely two genes will be inherited together.
•The same concept of studying how traitsare passed on was applied to develop
the first human genome map.
78

79. 2.Physical mapping
➢Physicallocationonthechromosome,relatingmoretowardsexactpositioningofgene
elements.
➢ItusesmolecularbiologytechniquestoexamineDNAmoleculesdirectlyinorderto
constructmapsshowingthepositionsofsequencefeatures,includinggenes.
➢bycontrast,alwaysgivethephysical,DNA-base-pairdistancesfromonelandmarkto
another.
➢Thedistancebetweenthelandmarksismeasureddirectlyaskilobases(Kb)ormegabases
(Mb).
➢Becausethedistanceisexpressedinphysicalunits,itismoreaccurateandreliablethan
centiMorgansusedingeneticmaps. 79

80. Cont’d…
❑Thereareaseveraldifferenttechniquesusedforphysicalmapping.Theseinclude:
➢A.Somaticcellhybridization
➢Thetechniqueofhybridproductionthroughthefusionofsomaticcellsunderinvitro
conditions,iscalledSomaticHybridization.
➢SomaticCellHybridizationinvolvesthefusionoftwodifferentcellstoproduceahybrid
whichistermedasHeterokaryon
➢Thefusionisusuallymediatedchemicallywithpolyethyleneglycol,whichaffectscell
membranes;orwithaninactivatedvirus,forexampletheSendaivirus,thatisabletofuse
tomorethanonecellatthesametime.
80

81. Cont’d…
➢ToisolatepurepopulationofHuman–MouseHeterokaryonaselectionprocedureisused
thatkillsboththeparentalcellsandthehomokaryonsbutallowsthehuman–mousehybrid
cellstosurviveandgrow.
➢Inthemediumcontainsadrugaminopterin,whichblocksthedenovopurineand
pyrimidinebiosyntheticpathwaysofcells.
➢However,thepresenceofhypoxanthineandthymidineallowthecellstoovercomethe
blockbysynthesizingtheirpurinesandpyrimidinesusingsalvagepathways.
81

82. Cont’d…
➢ForcellstogrowinHATmedium,twoenzymes,hypoxanthine-guaninephosphoribosyl
transferase(HGPRT)andthymidinekinase(TK)mustbefunctional.
➢MousecellisdeficientinTK(TK-)andahumancellisdeficientinHGPRT(HGPRT-)
➢Heterokaryons,hasnormalHGPRTgenederivedfromthemousegenomeandTKfrom
genefromthehumangenome.Thereforeonlyhybridcellgrows.
➢Thehybridsareinitiallyisolatedandtheirchromosomeanalyzed
82

83. Somatic cell hybridization…
83

84. B. Fluorescent in situ hybridization(FISH)
➢ThisusesfluorescentprobestodetectthelocationofDNAsequencesonchromosomes.
➢First,theprobesareprepared.Theseareshortsequencesofsingle-strandedDNA,that
matchtheDNAsequencethatthescientistwantstofind.
➢Theprobesarethenlabelledwithfluorescentdyebeforebeingmixedwiththe
chromosomeDNA,sothatitcanbindtoacomplementarystrandofDNAonthe
chromosome.
84

85. Cont’d…
➢ThefluorescenttagallowsthescientisttoseethelocationoftheDNA
sequenceonthechromosome.
➢Thesiteofhybridizationisvisualizedunderafluorescencemicroscope.The
siteofthegeneappearsasabrightspot.
➢This technique provides the most direct method of visualizing genes on
chromosomes.
85

86. Fluorescence in situ hybridization…
86

87. 3.5.Molecular Marker
➢Inbiology,onemaydefine‘markers’aslandmarkstocharacterizeindividualsor
differentiatebetweenanytwoindividuals
-forvariousresearchpurposesespeciallyplantandanimalbreeding,humangenetics
andforensics.
➢Markersarebasicallyusedtofindthelocationsofgenesofinterests.
➢Practically,anytraitwhichexhibitsvariationbetweenindividualscanbeusedasamarker.
❑Markersmaybe-
1.Morphological(phenotypic)–Anytraitvisibletonakedeye,example,plantheight,flower
color,fruitshapeandsoon.
87

88. Cont’d…
2.Biochemical–Allozymes(isozymes)whicharestructurallydifferentformsofthe
sameenzyme.
➢Isozymes:aspeciesofenzymethatexistsintotwoormorestructuralformswhichare
easilyidentifiedbytheiractivities.Examplebloodgrouping
➢3.Molecular–DNAmarkers.TheseDNAmarkersareessentiallylandmarksonthe
genomeofanindividual,
-whichcanbevisualizedthroughvarioustechniquestodetectdifferences
betweensmallstretchesofDNAofanytwoindividuals.
88

89. 3.5.1.Properties of Ideal Molecular Marker
➢Itmustbepolymorphic.
➢Co-dominantinheritancei.e.Itshoulddistinguishesthehomozygote'sand
heterozygote's.
➢Amarkershouldbeevenlyandfrequentlydistributedthroughoutthegenome.
➢Itshouldbeeasy,fastandcheaptodetect.
➢Itshouldbereproducible.
➢Stability,noinfluencesfromtheenvironment
89

90. 3.5.2.Type of molecular marker
✓Withrapidadvancementsinthefieldofmolecularbiologytoday,thereareagreat
numberoftheclassesofmolecularmarkersavailable.
✓Molecularmarkersmaybebroadlyclassifiedintothreecategoriesinthechronological
orderoftheirdevelopment.
❑FirstGenerationofmarkerswasthehybridizationbasedmarkers.
✓ThesearesocalledbecausetheDNAprofileisvisualizedthroughhybridizationof
DNAwithradioactivelylabeledprobesofknownsequence.
✓FirstGeneration:1980s
-BasedonDNA-DNAhybridizations,suchasRFLP.
90

91. Cont’d…
❑ThesecondgenerationofmarkerswerethePCRbasedmarkers,astheirassaywascarriedout
throughamplificationusingeitherarbitraryorsequencespecificprimers.
✓SecondGeneration:1990s
-BasedonPCR:Usingrandomprimers:RAPD
-Usingspecificprimers:SSR
-BasedonPCRandrestrictioncutting:AFLPandtheirmodification
❑Thethirdgenerationmarkersarethemostrecentones,calledasSNPs.Theirdetectionrequires
sequenceinformation.
✓WiththeadvancementinthefieldofDNAsequencing,SNPshavebecomeverypopularmolecular
markers.
-BasedonDNApointmutations(SNP),canbedetectedbysequencingetc.
91

92. ❑Commonly Used molecular marker for genome mapping are;
❖RFLP -Restriction fragment length polymorphism
•GeneticmarkersresultingfromthevariationorchangeinthelengthofdefinedDNA
fragmentsproducedbydigestionoftheDNAsamplewithrestrictionendonucleases.
•TheDNAfragmentisthenseparatedbyelectrophoresisandtransferredtoasolidsupport
(southernblot)
•HybridizewithradioactivelylabelledDNAprobe(s);detectbyautoradiography
92

93. Cont’d…
❖Microsatellitepolymorphism,SSRorSimplesequencesrepeat
•Microsatellites,SimpleSequenceRepeats(SSRs),orShortTandemRepeats(STRs),are
repeatingsequencesof1-6basepairsofDNA.
•SSRlengthpolymorphismsarecausedbydifferencesinthenumberofrepeats.
•SSRlociare“individuallyamplifiedbyPCRusingpairsofoligonucleotideprimers
specificto
-uniqueDNAsequencesflankingtheSSRsequence.
93

94. Cont’d…
94

95. Cont’d…
❖Single nucleotide polymorphisms(SNP’s)
➢Thistermreferstosinglenucleotidedifferencesbetweentwoorganismsthatareusually
foundthroughhighthroughputsequencingeffort
➢Arevariationinthenucleotidepresentataspecificpositiononachromosome
➢Occurinhighfrequency
-OneSNPoccursapproximatelyevery1,250basepairsinhumanDNA.
➢TheycanbeamplifiedwithPCRfortesting
95

96. Application of Molecular Marker
➢Phylogenetic studies
➢Trait identification and Mapping
➢DNA finger printing
➢Genetic diagnostics
➢Expression Profile Analysis
➢Gene mapping / Gene tagging
➢Marker Assisted Selection (MAS)
96

97. Individual assignment
•What is Genome analysis and annotation???
97

98. Chapter Three
Human Genome Project and Plant Genome program
98

99. 1.Human Genome Project(HGP)
➢TheHumanGenomeProject(HGP)wasaninternationalscientificresearchproject
that
-aimedtodeterminethecompletesequenceofnucleotidebasepairsthatmake
uphumanDNAandallthegenesitcontains.
➢Itremainstheworld'slargestcollaborativebiologicalproject.
➢Hundredsofresearchlaboratoriesandnationalresearchcentersfocusandcoordinate
thework.
➢TheProjectwascoordinatedby
✓TheNationalInstitutesofHealthandtheU.S.DepartmentofEnergy.
99

100. Cont’d..
➢Thesequencingofthehumangenomeinvolvedresearchersfrom20separateuniversitiesand
researchcentersacrosstheUnitedStates,UnitedKingdom,France,Germany,JapanandChina.
➢ThegroupsinthesecountriesbecameknownastheInternationalHumanGenomeSequencing
Consortium(IHGSC)
➢Theideawaspickedupin1984bytheUSgovernmentwhentheplanningstarted,theprojectwas
formallylaunchedin1990andwasdeclaredcompletein2003
➢TheHumanGenomeProjectoriginallyaimedtomapthenucleotidescontainedinahumangenome.
➢The"genome"ofanygivenindividualisunique;
-mappingthe"humangenome"involvedsequencingthegenomesofasmallnumberof
individualsandthenassemblingthesetogethertogetacompletesequenceforeachchromosome.
100

101. Cont’d…
101

102. Cont’d…
❑Millstones
➢1984ThebirthoftheHumanGenomeProject.
➢1990ProjectinitiatedasjointeffortofUSDepartmentofEnergyandtheNational
InstituteofHealth(NIH)inUSA.
➢1994,GeneticPrivacyAct:toregulatecollection,analysis,storageanduseof
DNAsamplesandgeneticinformationisproposed.
➢1996,WelcomeTrustjoinstheproject.
102

103. Cont’d…
➢1998,CeleraGenomics(aprivatecompanyfoundedbyCraigVenter)formedto
sequencemuchofthehumangenomein3years.
➢2000,Completionoftheworkingdraftoftheentirehumangenome.
➢2001,Analysisoftheworkingdraftarepublished.
➢2002workingdraftofmousegenomesequencewascompleted&published.
➢2003,HGPsequencingiscompletedandProjectisdeclaredfinishedtwoyearsaheadof
schedule
103

104. 1.1.Goals Of HGP
➢To identify all the genes in human DNA.
➢To determine the sequences of the 3.2 billion base pairs.
➢To store the information in databases;
➢To develop a genetic linkage map of human genome.
➢To obtain a physical map of human genome.
➢To develop technology for the management of human genome information.
➢To know the function of genes.
104

105. Cont’d…
➢Tofigureoutdifferencesbetweenindividualssothatgeneticdiseasescanbe
betterunderstoodandhopefullycured
-Note:Manythousandsofdiseasesofgeneticoriginhavebeenidentifiedin
whichthedefectisdistinguishedasamonogenic(single-gene)disorder.
➢Todetermineintron/exonstructureofallgenes
➢Revealnoncodingregulatorysequences
➢Identifypolymorphisms
➢Developmethodologyforothergenomes
105

106. Interesting fact or salient feature of human genome
➢The human genome is by far the most complex and largest genome.
➢The human genome contains over 3 billion nucleotide pairs and is
estimated to have about 30,000 genes .
➢Average gene consists of 3000 bases.
-But sizes of genes vary greatly, with the largest known human gene
encoding dystrophincontaining 2.5 million base pairs.
106

107. Cont’d…
➢Thefirstchromosometobecompletelydecodedwaschromosome22.
➢Chromosome1hasmostgenes(2968)andYchromosomehasthelowest(231).
➢Almostallnucleotidebasesareexactlythesameinallpeople.
-Genomesequencesofdifferentindividualsdifferforlessthan0.2%ofbasepairs.
➢Mostofthesedifferencesoccurintheformofsinglebasedifferencesinthesequence.
Thesesinglebasedifferencesarecalledsinglenucleotidepolymorphisms(SNPs).
107

108. Cont’d…
➢OneSNPoccursatevery~1,000bpofhumangenome.About85%ofall
differencesinhumanDNAsareduetoSNPs.
➢About1.5%ofthe3billionbparegenesthatcarryinformationusedbythecell
forsynthesisofproteins.
➢Mappingofhumangenomerequiresasetoflandmarks;
➢Mappinghumangenesiscriticallyimportantinmappingofdiseasegene,
-whichcreateanopportunityforresearcherstoisolatethegeneand
understandhowitcausesadisease.
108

109. 1.2.Issues Of Concern Ethical, Legal and Social issues of the Human
Genome Project
➢Fairnessintheuseofgeneticinformation.
➢Privacyandconfidentialityofgeneticinformation.
➢Psychologicalimpact,stigmatization,anddiscrimination
➢Reproductiveissues.
➢Clinicalissues.
➢Uncertaintiesassociatedwithgenetestsforsusceptibilitiesandcomplexconditions
109

110. 1.3.Application Of HGP
➢Thesequencingofthehumangenomeholdsbenefitsformanyfields,frommolecular
medicinetohumanevolution.
➢Helpsinidentifyingdiseasecausinggene.
➢Identificationofmutationslinkedtodifferentformsofcancer.
➢ThesequenceoftheDNAisstoredindatabasesavailabletoanyoneontheInternet.
➢Benefittedtheadvancementofforensicscience.
110

111. 1.4.Pros and cons of the Project
❖Pros
➢Successfully identifies where the genes of DNA are located in the body
➢Genetically modify foods
➢Make crops grow faster and more resistant to pesticides
➢Mapping can locate cancer cells and mental illnesses
❖Cons
➢15 years
➢$3-9 billion
➢Requires skill
➢Process is very difficult with lots of procedures
111

112. 2.Plant Genome Project
➢Announcedin2008,shortlyafterthehumanGenomesProject,thePlantGenomes
Projectisanother,similarhighlylarge-scalegenomicsendeavourtotakeadvantageof
thespeedandefficiencyofnext-generationDNAsequencing.
➢HeadedbyDr.GaneKa-ShuWongandDr.MichaelDeyholosoftheUniversityof
Alberta,
➢Theprojectaimsto;
-discoverallgenesandtheirfunctioninaparticularplantspecies.
-obtainthetranscriptome(expressedgenes)ofdifferentplantspecies.
112

113. Cont’d…
➢Asof2002,thenumberofclassifiedgreenplantspeciesisaround370,000.
➢However,thereareprobablymanythousandsmoreyetunclassified.Despitethisnumber,
veryfewofthesespecieshavedetailedDNAsequenceinformationtodate;125,426
speciesinGenBank.
➢Almostnoneoftheroughlyhalfmillionplantspeciesknowntohumanityhasbeen
touchedbygenomicsatanylevel“.
➢ThePlantGenomesProjectwillproducearoughlyincreaseinthenumberofspecieswith
availablebroadgenomesequence.
113

114. Cont’d…
❖ThegoalofthePlantGenomeResearchProgramis
➢Tosequencethewholegenome
➢todiscoverallthegenesandtheirfunctioninparticularplantspecies
➢toimproveplants(agronomic,horticultural,andforesttreespecies)
-bymakingcropsgrowfasterandmoreresistanttopesticides
114

115. Cont’d…
➢The Plant Genome Program is one program with two parts: National
Research Initiative (NRI) and Plant Genome Database (PGD).
➢The PGD is now a real and functioning information and data resource for
agricultural and other plant science genome researchers, and it is in the
public domain.
➢In crop plants, the genome mapping and sequencing has been completed in
two plant species; Arabidopsis and Rice.
115

116. Cont’d…
➢Arabidopsis thaliana was the first plant to be completely sequenced.
➢It is an important model plant for identifying genes and determining their
function of other plant species because Arabidopsis
-found in all over the world
-easy to grow
-basic similarity with the other crop
-simple genetic makeup
116

117. Cont’d…
➢Arabidopsisthalianagenomesequencingwascompletedinthelate2000
➢Otherplantgenomearebeingsequenced;e.g.Alfalfa,Maize,barley,
wheat,Sorghum,beet,cotton,potato,Soybean,Apple
117

118. 2.1.Relationship to HGP
➢Bothprojectsarelarge-scaleeffortstoobtaindetailedDNAsequenceinformationto
improveourunderstandingoftheorganisms,and
➢Bothprojectswillutilizenext-generationsequencingtofacilitateatimelycompletion.
➢Thegoalsofthetwoprojectsaresignificantlydifferent.
➢Thedifferenceincostcomesfromthetargetsequenceinthegenomes.
➢SincethePlantGenomesProjectwillonlybesequencingthetranscriptome,whereasthe
humanprojectwillsequenceasmuchofthegenomeasisdecidedfeasible.
118

119. Cont’d…
➢Thereisamuchloweramountofsequencingeffortneededinthismorespecific
approach.Whilethismeansthattherewillbelessoverallsequenceoutputrelativetothe
HumanGenomesProject,
➢Thenon-codingportionsofthegenomesexcludedinthePlantGenomesProjectarenot
importanttoitsgoalsliketheyaretothehumanproject.
➢ThemorefocusedapproachofthePlantGenomesProjectminimizescostwhilestill
achievingitsgoals.
119

120. 120

121. Chapter-4. Transcriptomics
121

122. 1.Transcriptomics
➢Thetranscriptomeisthecompletesetoftranscriptsinacellandtheirquantity,foraspecific
developmentalstageorphysiologicalcondition.Or
➢ThetranscriptomeisthesetofallRNAmolecules,including
▪mRNA,
▪rRNA,
▪tRNA, and other non-coding RNA produced in one or a population of cells.
➢Itisacollectionofallthegenetranscriptspresentinagivencell.
122

123. Cont’d…
➢Genomeswithinandacrossspeciesmightbeverysimilarhoweverthegenesthatare
expressedmakesthedifference.
➢Thetranscriptomeisdynamicwhilethegenomeisstatic
➢Onegenehasalwaysthesamesequence(exceptbymutation)butthesamegeneis
differentiallyexpressedatd/tratesind/tsituations.
➢Understandingthetranscriptomeisessentialforinterpretingthefunctionalelementsof
thegenomeandrevealingthemolecularconstituentsofcellsandtissues.
123

124. Cont’d…
➢ThestudyofTranscriptomeiscalledtranscriptomics,alsoreferredtoasexpression
profiling,examinestheexpressionlevelofmRNAsinagivencellpopulation,oftenusing
high-throughputtechniques.
➢UntilrecentyearsRNAisoverlookedcomparedtoDNAorproteinsconsignedtoamere
assistantroleintheflowofinformationfromgenestofunctioningmoleculesinlivingcells.
➢FewexcepthardcorefansexpectedRNAtoperformmoreinterestingrolessuchas
regulatinggeneexpression
➢ButthisstatusischangingasnewfunctionarebeingidentifiedforRNAmolecules
particularlyastheyhelptocontrolessentialeventsinallorganisms.
124

125. Why the study of RNA is so important??
❖RNAprofilingprovidecluesaboutthe
✓Functionaldifferencesbetweentissuesandcelltype
✓Functionandinteractionbetweengenes
✓Generegulationandregulatorysequences
✓Identificationofcandidategenesforanyprocessordisease
✓Expressedsequencesorgenesofgenome
125

126. How much RNA molecules are found in the cell/organism?
➢Atypicalbacteriumcontains0.05-0.10pgofRNAmakingupabout6ofitstotalweight
➢AmammaliancellbeingmuchlargercontainmoreRNA20-30pginallbutthis
representsonly1%ofthecellasawhole
➢ThereforebestwaytounderstandthismuchofRNAcontentofacellistodivideitinto
categoriesandsubcategoriesdependingonfunction
➢AccordinglytheprimarydivisioniscodingRNAandnoncodingRNA
126

127. 2.Types of RNA
❑2.1.ThecodingRNA(mRNAs)
➢MadeuponeclassofmoleculemRNAs,transcriptsofproteincodinggenes
➢Usedtoencodethesequenceofaminoacidsinapolypeptide
➢Maybepolycistronic(encode>twopolypeptides)inbacteriaandarchaea
➢Monocistronic(encodesinglepolypeptide)nearlyinalleukaryotes
➢Rarelymakeupmorethan4%ofthetotalRNAandareshortlivedbeingdegradedsoon
aftersynthesis
➢BacterialmRNAshavehalf-livesofnomorethanafewminutesand
-IneukaryotesmostmRNAsaredegradedwithinafewhoursaftersynthesis
127

128. Cont’d…
128

129. Cont’d…
❑2.2. Non coding RNAs
➢AlsocalledfunctionalRNAhaveessentialroleswithinthecell
➢FunctionalRNAscarryouttheiractivitiesinnucleicacidformarenottranslatedinto
protein.
❖ThereareseveraltypesoffunctionalRNAsthemostimportantare
✓RibosomalRNAs
✓TransferRNAs
✓SmallnuclearRNAs
✓MicroRNAs(miRNA)and
✓shortinterferingRNAs(siRNAs)
129

130. cont’d…
❖Ribosomal RNAs(rRNAs)
➢Foundinallorganismsandmostabundantmakingupover80%ofthetotal
RNAinactivelydividingbacteria
➢Arecomponentsofribosomesthestructureonwhichproteinsynthesis
takesplace
➢Alongwithnumerousproteinsthelargeandsmallribosomalsubunitsunite
fortranslationofmRNA
130

131. Cont’d…
❖Transfer RNAs (tRNAs)
➢Areinvolvedinproteinsynthesisandfoundinallorganisms
➢Contain15%oftotalRNA
➢CarriesaminoacidstoribosomesandbindstheretomRNAby
complementarybasepairinginordertodeposittheaminoacidstoelongatethe
polypeptide
➢ThefunctionoftRNAsistocarryaminoacidstotheribosomeandensurethat
theaminoacidsarelinkedtogether.
131

132. How mRNA ,tRNA and rRNA work together in translation to build protein
132

133. Cont’d…
❖Small nuclear RNAs [snRNAs)
•Alsocalledu-RNAbecausethesemoleculesarerichinuridineNucleotides.
•Foundineukaryoticnuclei,
-wheremultiplesnRNAsjoinwithnumerousproteinstoform
spliceosomesthatremoveintronsformprecursormRNAs.
•involvedinsplicingtheprimarytranscriptsofprotein–codinggenesinto
mRNAs
133

134. Cont’d…
134

135. ❖Small interfering RNA (siRNAs)
➢siRNAhasanimportantroleindefendingcellsagainstparasiticnucleotidesequences-
virusesandtransposons.
➢ThesiRNAspathwayisinitiatedbytheenzymeDicer,whichcleaveslongdouble–
strandedRNA(dsRNA)moleculesintoshortdoublestrandedfragmentsof~20
nucleotidesiRNAs.
➢EachsiRNAsisunwoundintotwosingle-strandedRNAs[ssRNAs],thepassenger
strandandguidestrand.
➢ThepassengerstrandisdegradedandtheguidestrandisincorporateintotheRNA-
inducedsilencingcomplex[RISC].
135

136. Cont’d…
•The most well –studied out come is post-transcriptional gene silencing,
-which occur when the guide strand pairs with a complementary sequence in a m
RNA molecule and induces cleavage
136

137. Cont’d…
137

138. Cont’d…
138

139. 3.Function of RNA
3.1. RNA as Genetic information storage/transfer;
➢AlthoughRNAdoesnotserveasthehereditaryinformationinmostcells,RNAdoes
holdthisfunctionformanyvirusesthatdonotcontainDNA
➢Thus,RNAclearlydoeshavetheadditionalcapacitytoserveasgeneticinformation.
3.2.RNAasstructuralcomponentofcell
➢CompositionofbothprokaryotesandeukaryotesribosomescontainrRNA
asstructuralcomponent.
139

140. 3.3. RNA as regulatory molecule
➢miRNAandsiRNAhavebeenshowntoregulatecertaingeneexpressionbybindingmRNA
preventtheirtranslationtoprotein.
3.4.RNAascatalystmolecule
➢Twoofthecell'smostimportantreactionsarecatalyzedbyRNA.
➢Theribozymecatalyzesspecificreactionsinasimilarwaytothatofproteinenzymes.Also
calledcatalyticRNA,ribozymesarefoundintheribosomewheretheyjoinaminoacids
togethertoformproteinchains.
140

141. 4.Methods for transcriptome or Gene expression analysis
•What is gene expression?
•Gene expression is the process by which the instructions in our DNA are
converted into a functional product, such as a protein.
•When the information stored in our DNA is converted into instructions for
making proteinsor other molecules, it is called gene expression.
•Gene expression is a tightly regulated process that allows a cell to respond
to its changing environment.
141

142. Cont’d…
➢It acts as both an on/off switch to control when proteins are made and also a
volume control that increases or decreases the amount of proteins made.
➢There are two key steps involved in making a protein, transcription and
translation.
❖Transcription
➢Transcription is when the DNA in a gene is copied to produce an RNA transcript
called messenger RNA(mRNA).
➢This is carried out by an enzyme called RNA polymerase which uses available
bases from the nucleus of the cell to form the mRNA.
142

143. Cont’d…
•RNAisachemicalsimilarinstructureandpropertiestoDNA,butitonlyhasasingle
strandofbasesandinsteadofthebasethymine(T),RNAhasabasecalleduracil(U).
143

144. Cont’d…
❖Translation
➢Translation occurs after the messenger RNA (mRNA) has carried the
transcribed ‘message’ from the DNA to protein-making factories in the cell,
called ribosomes.
➢The message carried by the mRNA is readby a carrier molecule called
transfer RNA (tRNA).
➢The mRNA is read three letters (a codon) at a time.
144

145. Cont’d…
➢Each codon specifies a particular amino acid. For example, the three bases ‘GGU’ code
for an amino acid called glycine.
➢As there are only 20 amino acids but 64 potential combinations of codon, more than one
codon can code for the same amino acid. For example, the codons ‘GGU’ and ‘GGC’
both code for glycine.
➢Each amino acid is attached specifically to its own tRNA molecule.
➢When the mRNA sequence is read, each tRNA molecule delivers its amino acid to the
ribosome and binds temporarily to the corresponding codon on the mRNA molecule.
145

146. Cont’d…
➢Once the tRNA is bound, it releases its amino acid and the adjacent amino acids all join
together into a long chain called a polypeptide.
➢This process continues until a protein is formed.
➢Proteins carry out most of the active functions of a cell.
146

147. Methods/Approach for transcriptome or Gene expression analysis
✓ExpressionSequenceTag(EST)
✓Serial/CapAnalysisofGeneExpression(SAGE/CAGE)
✓Microarray
✓RNA-Seq(RNASequancing)
147

148. 4.1.Expression Sequence Tag (EST)
➢Expressedsequencetags:-ESTsareshortsequencesofcDNAtypically200-
400nucleotidesinlength.
➢Obtainedfromeither5’endor3’endofcDNAinsertsofcDNAlibrary.
➢cDNALibrariescanbepreparedbyreversetranscriptionofmRNAbyusing
oligo(dT)primersthathybridizewiththepoly(A)tailofmRNAsandligationof
thecDNAstocloningvectors.
➢usedtoidentifytheunknowngenexpression,genomemapconstructionand
characterizationofexpressedgene
148

149. Cont’d…
➢Expressed Sequence Tags (ESTs)
➢ESTs are short (200–500 nucleotides) DNA sequences that can be used to identify a gene
that is being expressed in a cell at a particular time.
❖The Procedure:
➢Isolate the messenger RNA (mRNA) from a particular tissue (e.g., liver)
➢Treat it with reverse transcriptase. Reverse transcriptase is a DNA polymerase that uses
RNA as its template. Thus it is able to make genetic information flow in the reverse (RNA -
>DNA) of its normal direction (DNA -> RNA).
➢This produces complementary DNA (cDNA). Note that cDNA differs from the normal gene
in lacking the intron sequences.
➢Sequence 200–500 nucleotides at both the 5′ and 3′ ends of each cDNA.
➢Examine the database of the organism's genome to find a matching sequence.
149

150. Cont’d…
➢ESTwasusefulforunknowngeneidentificationbutcouldnotquantifyexpressedgenes.
➢AnothermajordrawbackofESTwassequencingasinglecDNAcopyatatime,makingit
lowthroughputandcostlymethod.
➢Withtheadventofnewtechnique,ESTisnolongerused,butESTlibrarieshavebeenthe
basisfordesigningofearlymicroarraygenechips.
150

151. 4.2.Serial/Cap Analysis of Gene Expression (SAGE/CAGE)
➢AnadvancementofESTwasserialanalysisofgeneexpression(SAGE)where
fragmentsweretaggedwhichallowedquantitationoftranscripts.
➢SAGEwasinventedin1995byDr.VictorVelculescufromJohnHopkins.
➢SAGEisapowerfultechniquedesignedfordirectquantitation(digitalanalysis)ofgene
expressionandalsoidentifiesnovelgeneexpressioninacellpopulation.
➢Expressionofgeneinacellvaryincertaincondition,soscientiststounderstandand
quantifywhichgeneisexpressedinwhatnumberincertainconditionuseSAGE
technique.
151

152. Cont’d…
➢Serialanalysisofgeneexpression(SAGE)isbasedontheprinciplethat
anoligonucleotidesequenceof9–10bpcanuniquelyidentifyagene
❖BasicprincipleunderlyingSAGEis:
(a)Simultaneousanalysisofthousandsofgenesbytaggingshortoligosequencesof9–10
bpat3′endforeachtranscript.
(b)Sequencetagsarelinkedtogether,thencloned,andsequenced.Serialandparallel
analysiscanbeperformedtogethertherebyincreasingthedataoutput.
(c)Quantitationisfinallydone,whereineachtagcountrepresentstheabundanceofa
particulartranscript.
152

153. Cont’d…
153

154. 4.3. Microarray based approach
➢Themostcommonlyusedglobalgeneexpressionprofilingmethodincurrentgenomics
researchistheDNAmicroarray-basedapproach
➢AlsotermedasDNAchips,genechips,DNAarrays,genearraysandbiochips.
➢DNAmicroarrayisoneofthemoleculardetectiontechniqueswhichisacollectionof
microscopiccharacteristics(commonlyDNA)affixedtoasolidsurface.
➢DNAmicroarraysaresolidsupportsusuallymadeupofglassorsiliconuponwhich
DNAisattachedinanorganizedpre-arrangedgriddesign.
154

155. Cont’d…
➢EachspotofDNA,termedasprobe,signifiesasinglegene.
➢DNAmicroarrayscanexaminetheexpressionoftensofthousandsofgenes
concurrently.
➢Thereare2typesofDNAmicroarrayi.e.cDNAbasedmicroarrayand
oligonucleotidebasedmicroarray.
❖PrincipleofDNAmicroarray:
➢DNAmicroarraytechnologywasoriginatedfromSouthernblotting,inwhich
fragmentedDNAisattachedtoasubstrateandthenprobedwithaknownDNA
sequence
155

156. Cont’d….
•DNAmicroarrayisbasedonprincipleofhybridizationbetweenthenucleicacidstrands.
•Complementarynucleicacidsequenceshavethecharacteristictospecificallypairtoeach
otherbytheformationofhydrogenbondsbetweencomplementarynucleotidebasepairs.
•UnknownsampleofDNAsequenceistermedassampleortargetandtheknown
sequenceofDNAmoleculeiscalledasprobe.
•Fluorescentdyesareusedforlabellingthesamples.
156

157. Cont’d…
➢Alargenumberofcomplementarybasepairsinnucleotidesequenceissuggestiveoftighter
non-covalentbondingbetweenthetwostrands.
➢Followingthewashingoffofnon-specificbondingsequences,onlystronglypairedstrands
willstayhybridized.
➢Thus,thefluorescentlabeledtargetsequencesthatpairstotheprobereleasesasignalthat
reliesonthestrengthofthehybridizationdetectedbythenumberofpairedbases,
hybridizationconditions,andwashingafterhybridization.
157

158. Cont’d…
➢Aftercompletionofthehybridization,thesurfaceofchipcanbeexaminedboth
qualitativelyandquantitativelyby
-useofautoradiography,laserscanning,fluorescencedetectiondevice,enzyme
detectionsystem.
❖RequirementsofDNAmicroarray:
•DNAchip
•Fluorescentlabelledtarget/sample
•Scanner
158

159. Steps involved in DNA microarray:
•The are four main steps involved in DNA microarray
1.Sample collection and Isolation of mRNA
2.Creation of labeled cDNA
3.Hybridization and washing
4.analysis
159

160. Cont’d…
1.Sample collection and isolating mRNA
•Asamplecanbeanycell/tissuethatwedesiretoconductourstudyon.
•Generally,2typesofsamplesarecollected,i.e.healthyandinfectedcells,for
comparingandobtainingtheresults.
•The extraction of RNA from a sample is performed by using a column or solvent like
phenol-chloroform.
•mRNA is isolated from the extracted RNA leaving behind rRNA and tRNA.
160

161. Cont’d…
➢As mRNA has a poly-A tail, column beads with poly-T tails are employed to bind mRNA.
➢Following the extraction, buffer is used to rinse the column in order to isolate mRNA from
the beads.
2.CreationoflabeledcDNA
➢Reverse transcription of mRNA yields cDNA.
➢Both the samples are then integrated with different fluorescent dyes for the production of
fluorescent cDNA strands which allows to differentiate the sample category of the cDNAs.
161

162. 3.Hybridization and washing
•ThelabeledcDNAsfromboththesamplesareplacedontheDNA
microarraywhichpermitsthehybridizationofeachcDNAtoits
complementarystrand.
•Thentheyarethoroughlywashedtoremoveunpairedsequences.
162

163. 4. Collection and Analysis
➢Microarrayscannerisusedtocollectthedata.
➢Thescannercontainsalaser,acomputerandacamera.Thelaserisresponsiblefor
excitingthefluorescenceofthecDNA,generatingsignals.
➢Thecamerarecordstheimagesproducedatthetimelaserscansthearray.
➢Thencomputerstoresthedataandyieldsresultsinstantly.Thedataarenow
analyzed.
➢Thedistinctintensityofthecolorsforeachspotdeterminesthecharacterofthe
geneinthatparticularspot.
163

164. Cont’d…
164

165. ❑Applications of DNA microarray technique
➢Drug discovery
➢Study of functional genomics
➢DNA sequencing
➢Gene expression profiling
➢Diagnostics and genetic engineering
➢Toxicological researches
➢Pharmacogenomics and theranostic
165

166. 4.4. RNA-Seq (RNA sequencing )
➢RNAsequencingisanadvancedtechniquewhichutilizesdeepsequencingapproachto
analyzethetranscriptome.
➢ThemajoradvantageofRNA-SeqovermicroarrayisthediscoveryofnovelRNAspecies.
➢RNAquantificationisdoneatsingle-baseresolutionandisacost-effectivehigh-throughput
analysisoftranscriptome.
➢RNA-Seqoffershighersensitivityanddynamicrangewhereabroadrangeofexpressionis
captured;microarrayexhibitssaturation,whileRNA-Seqatextremevaluesisinlinearscale.
➢RNAspliceeventscanbedetectedbyRNA-Seqwhilenotwithmicroarray
166

167. Cont’d…
❖Steps in RNA-seq:
•RNA isolation
•cDNA synthesis
•Library preparation
ofragmentation
oligation
•Sequencing
•Downstream applications
167

168. ❖RNA isolation:
➢The first step in the RNA sequencing is the isolation of total RNA, mRNA
or ncRNA for the experiment.
➢Isolating RNA is a tedious process in comparison with DNA isolation. The
RNA can be breakdown easily. Also, the chance of contamination is high in
RNA isolation.
➢Thus extra care must be taken while isolating RNA and expert hand in
required to do so.
➢High yield pure RNA is must require for RNA sequencing.
168

169. Cont’d…
➢Note: for isolating mRNA from the total RNA pool, one additional step is
required, using the oligo dT specific column in the purification or final
step of elution, total mRNA can be isolated from the rest of RNAs.
❖Reverse transcriptase PCR:
➢Another innovative set up for RNA sequencing is to do reverse transcriptase PCR in
which the RNA is reverse transcribed into DNA.
➢Using a special type of polymerase known as DNA reverse transcriptase, the cDNA is
synthesized from the mRNA.
169

170. Cont’d…
➢SecondstrandcDNAsynthesis:
➢AfterthecDNAsynthesizedfromthemRNA,thesecondstrandDNA
synthesisismustrequired.Forthat,PCRisperformedusingthenormalTaq
DNApolymeraseinconventionalPCR.
➢ThePolymeraseaddsdNTPstothegrowingDNAstrandusingtheprimerset.
170

171. ❖Library preparation
➢ThefirststepinthelibrarypreparationorNGSlibrarypreparationstarts
withthefragmentations.
➢UsingthespecialtypeofrestrictionendonucleaseswholesetofdscDNAis
fragmentedfortheNGS.
➢(NB)Thelibrarypreparationvariesfromplatformtoplatformsassome
fragmentsmRNAbeforeperformingreversetranscriptasewhilesome
performitlaterafterthepreparationofcDNA.
171

172. Cont’d…
➢Inthelaststepofthelibrarypreparation,theendsareligatedwiththeadaptersequences
andamplifiedforrepairingtheends.
➢Adaptersarethenaddedtoeachendofthefragments.Theseadapterscontainfunctional
elementswhichpermitsequencing;forexample,theamplificationelementandtheprimary
sequencingsite.
Librarypurification:
➢TheentirelibraryispurifiedusingthereadytouseDNApurificationkit.
➢Inthelaststep,itisverycrucialtopurifytheentirefragmentlibraryforthatthe
concentrationofthelibraryisassessedusingthequantitativePCRorbioanalyzer.
172

173. Cont’d…
•In the next step, the sample of fragmented DNA is sent for sequencing.
•The fragmentation method is a key aspect of sequencing library
construction.
•Fragmentation may be achieved by chemical hydrolysis, nebulization,
sonication….
173

174. ❖DNA sequencing:
➢Now the sample is sequenced in the high throughput NGS machine which
reads the sequence as well as quantifies the nucleic acid as well.
➢The chemistry behind sequencing the cDNA is depends on which platforms
we are using, although, the widely used method is the use of fluorescence
chemistry
➢In the sequencer, individual fragments are “read” separately and sequenced.
➢The final data are sent to the bioinformatics lab for post-sequencing analysis.
174

175. Cont’d…
175

176. 4.4.1.Different RNA sequencing methods:
❖Wholetranscriptomesequencing:Thewholetranscriptomeofacellortissue-all
RNAs(mRNA,tRNA,rRNA,sncRNA,microRNAandsiRNA)aresequencedand
quantified.
❖TargetRNAsequencing:Agene-specific,clusterofgene-specific,apathway-
specificordisease-relatedtranscriptomesaresequencedinthetarget-specificRNA
sequencing.
❖SmallRNAsequencing:OneofthemostemergingtechniqueinRNA-sequencingis
tostudythesmallernon-codingRNAofacellbecausethoseareassociatedwithso
manyfunctionsinagenome.
176

177. Cont’d…
❖mRNAsequencing:
➢SequencetheentiremRNAtranscriptusingpoly(A)tailselectionusedforgeneexpression
studies.
➢UsingmRNAsequencingknownaswellasnoveltranscriptalterationcanbedetected.
Advantages of RNA sequencing:
➢OneofthepivotaladvantagesofRNAsequencingisthenoneedforpriorsequence
information.
➢Astheentireprocessisnotbasedontheprobe-basedchemistry,priorsequence
informationfordesigningtheprobeisnotrequiredhere.
177

178. Cont’d…
➢Moreaccurateandsensitivegeneexpressionstudycanbedone.
➢Eventhoughnoreferencesequenceinformationisavailable,RNAsequencingcanbeapplied
foranyspecies.
➢RNA-seqcantelluswhichgenesareturnedoninacell,whattheirlevelofexpressionis,and
atwhattimestheyareactivatedorshutoff.
➢Thisallowsscientiststomoredeeplyunderstandthebiologyofacellandassesschangesthat
mayindicatedisease.
➢Conclusivelywecansay,thattheRNAseqmethodismoreadvancedandaccurateas
comparedtothemicroarray.
178

179. Types of RNA-seq technologies:
➢Roche454-thefirstNGSTechnologyforRNASeqwasintroducedbyRochein2004,
179

180. Cont’d…
➢TheIlluminaplatform-introducedin2006,providinghighlyaccuratesequencingwitha
lowerrorrate,evenwithinrepetitivesequenceregions.
➢SOLID-Sequencing by oligo ligation and detection (SOLID) technology, released in 2007,
uses a sequencing chemistry catalyzed by DNA ligase and produces an average read length
of 75bp.
180

181. Cont’d…
181

182. Chapter five
182

183. 1.Introduction:
Whatisproteomics?
➢Theidentification,characterizationandquantificationofallproteinsinvolvedina
particularpathway,organelle,cell,tissue,organororganism
-thatcanbestudiedinconcerttoprovideaccurateandcomprehensivedataabout
thatsystem.”OR
➢Thestudyoftheexpression,location,interaction,functionandstructureofalltheproteins
inagivencellororganism
183

184. Cont’d…
Proteomicsismuchmorecomplicatedthangenomicsmostlybecausewhileanorganism's
genomeismoreorlessconstant,theproteomediffersfromcelltocellandfromtimeto
time.
➢Thisisbecausedistinctgenesareexpressedindistinctcelltypes.
➢Thismeansthateventhebasicsetofproteinswhichareproducedinacellneedstobe
determined
➢Proteomicsconfirmsthepresenceoftheproteinandprovidesadirectmeasureofthe
quantitypresent.
➢Generallyisproteomicsisthestudyofproteome.
184

185. What is proteome?
➢Proteomeistheentiresetofproteinsexpressedbyagenome,cell,tissueororganism.
➢Morespecifically,itisthesetofexpressedproteinsinagiventypeofcellsoranorganism
atagiventimeunderdefinedconditions.
➢Thetermisablendofproteinsandgenome
➢Differenttypesofcellsmakedifferentproteins,
-sotheproteomeofonecellwillbedifferentfromtheproteomeofanother
185

186. Cont’d..
➢The proteome is extremely dynamic, and minor alterations in the external or internal
environment can modify proteome function
➢By comparing protein sequences and structures, scientists can classify many proteins in an
organism’s proteome and deduce their functions by homology with proteins of known
function.
➢In the past protein determination was done by mRNA analysis, but this was found not to
correlate with protein content.
186

187. Cont’d…
➢Some times there is lack of correlation between mRNA and protein abundances. Why ???
➢One of the reasons for this is the existence of control mechanisms at the ribosomes, where
mRNA is translated to peptides.
➢Translational control allows the cell to select only certain mRNAs for translation and block
others.
➢The selection is often dependent on environmental conditions, so this mechanism allows
for physiological adaptation on the level of the proteome, even though the transcriptome
remains the same
187

188. Cont’d…
➢It is now known that mRNA is not always translated into protein, and the amount of
protein produced for a given amount of mRNA depends on the gene it is transcribed
from and on the current physiological state of the cell.
➢Another issue is post-translational modification or protein processing, processes that
can greatly affect the function of a protein.
➢Not only does the translation from mRNA cause differences, many proteins are also
subjected to a wide variety of chemical modifications after translation.
➢Many of these post-translational modifications are critical to the protein's function
188

189. Cont’d…
➢So,inamolecularbiologycontext,thelivingcellcanonlybeunderstoodfullyby
consideringgenome,transcriptome,andproteometogether.
➢Thebehaviorofgeneproductsisdifficultorimpossibletopredictfromgenesequence.
➢Evenifgeneistranscribed,itsexpressionmayberegulatedatleveloftranslation.
➢Proteinproductsaresubjectedtofurthercontrolbyposttranslationalmodifications.
➢So,proteomicsisafieldthatpromisestobridgethegapbetweengenomeandcellular
behavior.
189

190. 190

191. ❖What is protein ??
❖Discuss different types of protein based on their function and structure???
191

192. 2.Protein
•Proteins are nitrogenous organic compounds of high molecular weight which
play a vital or prime role in living organisms. They are made up of a-amino
acids.
❖Amino Acids
•Amino acids are the monomers that make up proteins.
•Each amino acid has the same fundamental structure, which consists of a
central carbon atom, also known as the alpha (α) carbon, bonded to an amino
group (NH2), a carboxyl group (COOH), and to a hydrogen atom
192

193. Cont’d…
193

194. Cont’d…
➢Everyaminoacidalsohasanotheratomorgroupofatomsbondedtothecentralatom
knownastheRgroup.
➢ThisRgroup,orsidechain,giveseachaminoacidproteinsspecificcharacteristics,
includingsize,polarity,andpH.
➢Thereare20aminoacidspresentinproteins,eachwithaspecificRgrouporsidechain.
➢Tenoftheseareconsideredessentialaminoacidsinhumansbecausethehumanbody
cannotproducethemandtheymustbeobtainedfromthediet.
194

195. ➢The sequence and the number of amino acids ultimately determine the protein’s
shape, size, and function.
➢Each amino acid is attached to another amino acid by a covalent bond, known as a
peptide bond.
➢When two amino acids are covalently attached by a peptide bond, the carboxyl
group of one amino acid and the amino group of the incoming amino acid combine
and release a molecule of water.
➢Any reaction that combines two monomers in a reaction that generates H2O as one
of the products is known as a dehydration reaction.
195

196. Cont’d…
196

197. 2.1.Types of protein
❖Protein can be grouped into several broad functional classes:
➢Structural proteins, which provide structural rigidity to the cell;
➢Transport proteins, which control the flow of materials across cellular
membranes;
➢Regulatory proteins, which act as sensors and switches to control protein
activity and gene function
➢Signaling proteins, including cell surface receptors and other proteins that
transmit external signals to the cell interior; and
➢Motor proteins, which cause motion.
197

198. Cont’d
198

199. 2.2.Function of Protein
➢Proteins perform essential functions throughout the systems of the human body.
These long chains of amino acids are critically important for:
✓catalyzing chemical reactions
✓synthesizing and repairing DNA
✓transporting materials across the cell
✓receiving and sending chemical signals
✓responding to stimuli
✓providing structural support
199

200. Enzymes
➢Enzymes are proteins that catalyze biochemical reactions, which otherwise would not take
place.
➢These enzymes are essential for chemical processes like digestion and cellular metabolism.
Without enzymes, most physiological processes would proceed so slowly (or not at all) that
life could not exist.
➢Because form determines function, each enzyme is specific to its substrates.
➢The substrates are the reactants that undergo the chemical reaction catalyzed by the enzyme
200

201. Cont’d…
➢The location where substrates bind to or interact with the enzyme is known as
the active site, because that is the site where the chemistry occurs
➢There are two basic classes of enzymes:
➢Catabolic enzymes: enzymes that break down their substrate
➢Anabolic enzymes: enzymes that build more complex molecules from their
substrates
201

202. Cont’d…
➢Enzymesareessentialfordigestion:theprocessofbreakinglargerfoodmolecules
downintosubunitssmallenoughtodiffusethroughacellmembraneandtobe
usedbythecell.
➢Theseenzymesincludeamylase,whichcatalyzesthedigestioncarbohydratesin
themouthandsmallintestine;
➢pepsin,whichcatalyzesthedigestionofproteinsinthestomach;
➢lipase,whichcatalyzesreactionsneedtoemulsifyfatsinthesmallintestine;
➢andtrypsin,whichcatalyzesthefurtherdigestionofproteinsinthesmall
intestine.
202

203. Cont’d…
➢Enzymes are also essential for biosynthesis: the process of making new, complex
molecules from the smaller subunits that are provided to or generated by the cell.
➢These biosynthetic enzymes include DNA Polymerase, which catalyzes the
synthesis of new strands of the genetic material before cell division;
➢fatty acid synthetase, which the synthesis of new fatty acids for fat or membrane
lipid formation;
➢and components of the ribosome, which catalyzes the formation of new
polypeptides from amino acid monomers.
203

204. Hormones
➢Someproteinsfunctionaschemical-signalingmoleculescalledhormones.
➢Theseproteinsaresecretedbyendocrinecellsthatacttocontrolorregulate
specificphysiologicalprocesses,whichincludegrowth,development,metabolism,
andreproduction.
➢Forexample,insulinisaproteinhormonethathelpstoregulatebloodglucose
levels.
➢Otherproteinsactasreceptorstodetecttheconcentrationsofchemicalsandsend
signalstorespond.
204

205. ❖Other Protein Functions
➢Proteinsperformessentialfunctionsthroughoutthesystemsofthehumanbody.
➢Intherespiratorysystem,hemoglobin(composedoffourproteinsubunits)transports
oxygenforuseincellularmetabolism.
➢Additionalproteinsinthebloodplasmaandlymphcarrynutrientsandmetabolicwaste
productsthroughoutthebody.
➢Theproteinsactinandtubulinformcellularstructures,whilekeratinformsthestructural
supportforthedeadcellsthatbecomefingernailsandhair.
➢Antibodies,alsocalledimmunoglobins,helprecognizeanddestroyforeignpathogensinthe
immunesystem.
205

206. 2.3. Protein Structure
➢Proteins are the polymers of amino acids.
➢Individual amino acids (residues) are joined by peptide bonds to form the linear polypeptide
chain.
➢This linear polypeptide chain is folded into specific structural conformations or simply
‘structure’.
➢A protein can have up to four levels of structural conformations.
206

207. Cont’d…
A protein can have Four levels of structural organization:
•They are designated as:
1.Primary Structure
2.Secondary Structure
3.Tertiary Structure
4.Quaternary Structure
207

208. 208

209. 3.Proteomicstechniques
➢Moleculartechniques
➢Separationtechniques
➢ProteinIdentificationtechniques
➢ProteinStructuretechniques
209

210. ❖3.1 Molecular techniques
•I.DNA Microarrays or Gene Chips
•II. Northern/Southern Blotting
•III. Serial Analysis of Gene Expression (SAGE)
210

211. 3.2.Separation techniques
❖Gel Electrophoresis
✓SDS-PAGE
✓2D Gel Electrophoresis
➢Gel electrophoresis: it is a technique used for the separation of DNA, RNA,
and protein molecules according to their size and electrical current using
electric current on a gel.
➢Therefore, gel electrophoresis is used for the separation of proteins on the
basis of their net negative charge and their size.
211

212. 3.2.1.Protein Electrophoresis
➢Proteinelectrophoresisislaboratorymethodthatseparateamixtureofproteinbasedon
theirchargeandsize
➢UsePolyacrylamideasgel,itisacross-linkedpolymerofacrylamide
➢Havesmallerporesthanagarose,thereforehighdegreeofresolvingpower.
➢CanseparateDNAfragmentswhichrangeinsizefrom10-500bp.
➢DNAfragmentswhichdifferinsizebyonenucleotidecanbeseparatedfromeach
other.
212

213. 3.2.2.SDS-PAGE
➢SDS-PAGE(sodium dodecylsulphate-polyacrylamide gel electrophoresis)
➢The purpose of this method is to separate proteins according to their size,
and no other physical feature.
➢In order to understand how this works, we have to understand the two halves
of the name: SDSand PAGE
213

214. Cont’d…
➢Sincewearetryingtoseparatemanydifferentproteinmoleculesofavarietyofshapesand
sizes,
✓wefirstwanttogetthemtobelinear
✓nolongerhaveanysecondary,tertiaryorquaternarystructure(i.e.wewantthemtohave
thesamelinearshape).
➢Notonlythemassbutalsotheshapeofanobjectwilldeterminehowwellitcanmove
throughandenvironment.
➢Soweneedawaytoconvertallproteinstothesameshape-weuseSDS.
214

215. Cont’d…
➢SDS(sodiumdodecylsulfate)isadetergentthatcandissolvehydrophobicmoleculesbut
alsohasanegativecharge(sulfate)attachedtoit.
➢IfSDSisaddedtoproteins,theywillbesoluablizedbythedetergent,plusallthe
proteinswillbecoveredwithmanynegativecharges.
➢Asampleofprotein,oftenfreshlyisolatedandunpurified,isboiledinthedetergent
sodiumdodecylsulfateandbeta-mercaptoethanol.
➢Themercaptoethanolreducesdisulfidebonds
➢Thedetergentdisruptssecondaryandtertiarystructure
215

216. Cont’d…
➢Theendresulthastwoimportantfeatures:
✓allproteinscontainonlyprimarystructureand
✓allproteinshavealargenegativechargewhichmeanstheywillallmigratetowards
thepositivepolewhenplacedinanelectricfield.
➢Theymigratethroughageltowardsthepositivepoleatarateproportionaltotheir
linearsize
✓Molecularweightswithrespecttosizemarkersmaythenbedetermined
➢ThebandoftheproteininthegelstainedbyCoomassieBrilliantBlueorsilver
staining.
216

217. Movement of Proteins in Gel…..
217

218. 3.2.3 2D Gel electrophoresis
➢Twodimensionalgelelectrophoresisis(2DE)isaclassicalmethodtoseparateprotein
ontheirbaseofcharge(isoelectricfocusing,IEF)andoftheirsize(SDS-PAGE).
218

219. Cont’d…
219

220. Cont’d…
220

221. Cont’d…
221

222. 3.3. Chromatography
➢Theseparationofcomponentsinamixturethatinvolvespassingthemixturedissolvedina
"mobilephase"throughastationaryphase,
-whichseparatestheanalytetobemeasuredfromothermoleculesinthemixture
basedondifferentialpartitioningbetweenthemobileandstationaryphases.Itincludes…
➢High performance liquid chromatography
➢Size, exclusion chromatography-separates proteins based on their molecular size
➢Ion exchange chromatography-separates proteins based on charge
➢Reverse phase chromatography
222

223. cont’d…
➢affinity chromatography employs reversible interactions between specific
affinity ligands and their target proteins
➢These methods can be used to purify and identify proteins of interest, as
well as to prepare proteins for further analysis
223

224. 3.4. Protein Microarray(protein chip)
➢They are modeled after DNA microarrays, in 2000 at Harvard University
➢The success of DNA microarrays in large-scale genomic experiments
inspired researchers to develop similar technology to enable large-scale,
high-throughput proteomic experiments.
➢Protein chips enable researchers to quickly and easily survey the entire
proteome of a cell within an organism.
224

225. Types of protein Microarrays
225

226. 3.4.1. Analytical microarrays
•Different types of ligands, including antibodies, antigens, DNA or RNA aptamers,
carbohydrates or small molecules, with high affinity and specificity, are spotted down onto a
derivatized surface.
•Protein samples from two biological states to be compared are separately labelled with red
or green fluorescent dyes, mixed, and incubated with the chips. Spots in red or green color
identify an excess of proteins from one state over the other.
•These types of microarrays can be used to monitor differential expression profiles and for
clinical diagnostics.
•Examples include profiling responses to environmental stress and healthy versus disease
tissues
226

227. ➢Usedtounderstand:expressionlevels,bindingaffinitiesandspecificities,responseof
thecellstoaparticularfactor,identificationandprofilingofdiseasedtissues.
227

228. 3.4.2. Functional protein microarrays (target protein arrays)
➢Immobilisedpurified proteins are used to:
-identify protein-protein/DNA/RNA
-assay enzymatic activity.
➢They differ from analytical arrays in that they contain full-length functional
proteins.
228

229. Cont’d…
➢Functional protein microarrays differ from analytical arrays in that
functional protein arrays are composed of arrays containing full-length
functional proteins or protein domains.
➢These protein chips are used to study the biochemical activities of an
entire proteome in a single experiment.
➢They are used to study numerous protein interactions, such as protein-
protein, protein-DNA, protein-RNA, protein-phospholipid, and protein-
small molecule interactions
229

230. 3.4.3 Reverse phase protein microarray (RPPA)
➢InRPPA,cellsareisolatedfromvariouscomplextissuesofinterestandare
lysed.
➢Thelysateisarrayedontoanitrocelluloseslideusingacontactpin
microarrayer.
➢Theslidesarethenprobedwithantibodiesagainstthetargetproteinof
interest,andtheantibodiesaretypicallydetectedwithchemiluminescent,
fluorescent,orcolorimetricassays.
230

231. Cont’d…
•RPPAsallowforthedeterminationofthepresenceofalteredproteinsthatmaybe
theresultofdisease.
231

232. 3.5.Protein Identification techniques
I. Edman sequencing
➢Edman Degradation is a method of sequencing amino acids in a peptide. Here,
the amino-terminal residue is labeled and cleaved from the peptide without
disrupting the peptide bonds between other amino acid residues
II. Mass spectroscopy
➢is an analytical technique that measures the mass-to-charge ratio of charged particles
for determining masses of particles and the elemental composition of a sample of
molecules
232

233. 3.6.Protein Structure techniques
I.NMR(Nuclear magnetic resonance spectroscopy)
II.X-ray crystallography
233

234. NMR(Nuclear magnetic resonance spectroscopy)
•It is based on the measurement of absorption of electromagnetic radiation in the
form of radio-frequency
•It is a technique which exploits the magnetic properties of certain nuclei to study
physical, chemical, and biological properties of matter
•Compared to MS, larger amounts of sample are needed, but non destructive
•Protein folding studies can be done by monitoring NMR spectra
•NMR is very efficient in mapping interactions with other molecules
•The upper weight limit for NMR structure determination is -30kDa
234

235. X-ray crystallography
•is a technique for examining the structure of a regular crystal.
•It only works on substances that can be crystallized.
•An x-ray source is aimed at the crystal, and the diffraction pattern that is created
as the x-rays strike the crystal are studied.
•Above 30kDa, X-ray is the only technique to solve the structure of proteins
Limitations
•Crystallization required, potential crystal packing influence the structure
•Can not be used with very flexible molecules
235

236. 3.7.Types of Proteomics
1. Expression proteomics
➢Analysis of protein expression at larger scale
➢Quantitative determination protein expression in different cell, tissue and organ
➢Helps to identify target proteins in a particular sample, and those proteins
differentially expressed in related samples such as diseased vs. healthy tissue.
➢If a protein is found only in a diseased sample then it can be useful for drug
target or diagnostic marker
➢Proteins with the same or similar expression profiles may also be functionally
related.
236

237. 2. Structural proteomics
–Analysis of protein structures at large-scale
–It compares protein structures and helps to identify functions of newly
discovered genes
–Helps to understand where drugs bind to proteins and also show where
proteins interact with each other
–This understanding is achieved using different technologies such as X-ray
crystallography and NMR spectroscopy.
237

238. 3.Functionalproteomics
➢Itreferstotheuseofproteomicstechniquestoanalyzethecharacteristicsof
molecularprotein-networkinvolvedinlivingcell.
➢Whichisidentificationof
✓Protein-proteininteraction
✓Protein-DNA
✓Protein-RNAinteraction
238

239. 3.8.Application of proteomics
➢Mining:identificationofproteins(catalogtheproteome)
➢Protein-expressionprofile:identificationofproteininterestinaparticularstate
ofthetissue/organism
➢Protein-networkmapping:proteininteractionsinlivingsystems
➢Mappingofproteinmodifications:howandwhereproteinsaremodified.
➢BiomarkersEg:ELISA&immunohistochemicalstainingetc..
➢Alzheimer'sdisease,HeartdiseaseandCancerouscells
239

240. CHAPTER 6 -METABOLOMICS
240

241. 1.Introduction: what is metabolomics?
➢Metabolomicsisanewlyemergingfieldofresearchconcernedwiththehigh-throughput
identificationandquantificationofthesmallmoleculemetabolitesinthemetabolome.
OR
➢Metabolomicsisthe"systematicstudyoftheuniquechemicalfingerprintsthat
specificcellularprocessesleavebehind"
-specifically,thestudyoftheirsmall-moleculemetaboliteprofiles.
241

242. Con…
➢WhatisaMetabolome?
➢Metabolomereferstothecompletesetofsmallmoleculemetabolites
-(suchasmetabolicintermediates,hormonesandothersignalingmolecules,and
secondarymetabolites)tobefoundwithinabiologicalsample(cell,organ,tissueor
organism)
➢Includesendogenousandexogenousmoleculesaswellastransientoreventheoretical
molecules
➢Metabolomesizeisalwaysill-defined
242

243. Con…
❖WhatisaMetabolite?
➢Metabolitesaretheintermediatesandproductsofmetabolism.Thetermmetaboliteis
usuallyrestrictedtosmallmolecules.
➢AnyorganicmoleculedetectableinthebodywithaMW<1500Da
➢Unlikegenesandproteins,serveasdirectsignaturesofbiochemicalactivityandare
mucheasiertocorrelatewithphenotype.
➢Includespeptides,oligonucleotides,sugars,nucelosides,organicacids,ketones,aldehydes,
amines,aminoacids,lipids,steroids,alkaloids,foods,foodadditives,toxins,pollutants,
drugsanddrugmetabolites
➢Includeshuman&microbialproducts
243

244. Why is Metabolomics Important?
1.Because it deals with Small Molecules
➢>95%ofalldiagnosticclinicalassaystestforsmallmolecules
➢89%ofallknowndrugsaresmallmolecules
➢50%ofalldrugsarederivedfrompre-existingmetabolites
➢30%ofidentifiedgeneticdisordersinvolvediseasesofsmallmoleculemetabolism
➢Smallmoleculesserveascofactorsandsignalingmoleculesto1000’sofproteins
244

245. Con…
245

246. Con…
3.TheMetabolomeisConnectedtoallother“Omes”
25,000 Genes
7500 Enzymes
8000
Chemicals
Metabolomics
Proteomics
Genomics
Environmental Influence
246

247. Con…
4.TheMetabolomeisConnectedtoAllOther“Omes”
➢Smallmolecules(i.e.AMP,CMP,GMP,TMP)aretheprimaryconstituentsofthe
genome&transcriptome
➢Smallmolecules(i.e.the20aminoacids)aretheprimaryconstituentsoftheproteome
➢Smallmolecules(i.e.lipids)givecellstheirshape,form,integrityandstructure
➢Smallmolecules(sugars,lipids,AAs,ATP)arethesourceofallcellularenergy
➢Smallmoleculesserveascofactorsandsignalingmoleculesforboththeproteomeand
thegenome
247

248. Con…
➢Thelevelofametaboliteisdeterminedbytheactivitiesofalltheenzymesofallthe
pathwaysthatinvolvethatmetabolite,andbyeffectorsthatactontheseenzymes.
➢Inpractice,therefore,metabolitelevelschangeaccordingtodevelopmental,
physiological,andpathologicalstates.
➢Metabolomicsanalysiscanpowerfullycomplementtranscriptomicsandproteomics.
➢Transcriptomesorproteomesareveryinadequatemonitorsofcellfunctionbecausethere
isnosimplerelationshipbetweenmRNAorproteinlevelsandmetabolism.
➢Metabolomicsanalysisconductedbydifferentmethodortechniques.
248

249. 2. Metabolomics Methods
➢SeparationTechniques
✓GasChromatography(GC)*
✓HighPerformanceLiquidChromatography(HPLC)*
➢DetectionTechniques
✓NuclearMagneticResonanceSpectroscopy(NMR)
✓MassSpectrometry(MS)
249

250. 2.1.Separation techniques
2.1.1.Chromatography
Theseparationofcomponentsinamixturethatinvolvespassingthemixturedissolvedina
"mobilephase"throughastationaryphase,whichseparatestheanalytetobemeasuredfrom
othermoleculesinthemixturebasedondifferentialpartitioningbetweenthemobileand
stationaryphases.
➢Chromatographytechniquesworksbasedontheprinciplethatthecomponentofthemixture
distributedunequallybetweentwoimmisciblephaseoneofthemiscalledthemobilephase,
whichisusuallyliquid
➢Thisliquidallowedtoflowoverthestationaryphase,whichisusuallysolidbutcanbeliquid.
250

251. Con…
➢Themixtureconsistoftheproductsthathavedifferentdegreeofattractiontothemobile
andstationaryphase.
➢Whenamixtureisplacedintomobilephaseandthemobilephaseallowedtomovealong
thestationaryphase
➢thesubstrateofthemixturethatbondstronglywiththestationaryphasewillmove
slowly
-ontheotherhandthesubstratethatbondlessstronglytostationaryphasewillmovequickerwith
themobilephase
251

252. 2.1.2.Types of chromatography
❖Manychromatographictechniqueexistandtheyarecategorizedbythenatureofmobile
andstationaryphase;
1.Liquid-solidphase
ExampleHPLC,thinlayer,column…
2.Gas-liquidphase
ExampleGaschromatography
252

253. ❖High Pressure (Performance) Liquid Chromatography -HPLC
➢Developedin1970’s
➢Useshighpressurespump(40MP)attachedtothecolumn
➢Thecolumnfilledwithadsorbentmaterialwhichhasaverysmallparticlesizethat
givehighsurfaceareatothesampleasresultefficiencyofseparationgivinghigh
resolution
➢Allowsseparationofmanytypesofpolarandnonpolarcompounds
253

254. Con…
❖Components of HPLC;
➢Column-
✓stainless still can with stand pressure upto50MP
✓The length of the column 5-25cm
✓4-5mm in diameter
✓Flow rate 1-3ml/m
➢Stationary phase –
✓adsorbentmaterialverysmallparticlesizewhichischemicallymodifiedsilica,
divinylbenzeneetc.
254

255. Con…
➢Mobile phase –
✓a mixture of solvent can be used as mobile phase
✓Mobile phase solvent can be polar non polar depends on the sample
➢Detector
✓attachedtotheendofthecolumn
✓Detectinformationfromthestationaryphaseanddelivertocomputer
through
✓DifferenttypesofdetectorUVdetector,IRdetector…
255

256. ❑Types of HPLC
➢Normalphase–forseparationofnon-polarmolecules(polarstationaryphase,non-
polar/organicmobilephase)
➢Reversedphase–forseparationofnon-polarmolecules(non-polarstationaryphase,
polarmobilephase)
➢Sizeexclusionchromatography-separationbasedonsize,smallermoleculeenter
theporeofthebeadandlargemoleculeremainoutside
➢Ionexchangechromatography-separationoccurbasedonmolecularcharge
256

257. Con…
257

258. ❖Gas chromatography
➢Involvesasamplebeingvaporizedtoagasandinjectedintoacolumn
➢Usedtoseparatevolatilecomponds
➢Sampleistransportedthroughthecolumnbyaninertgasmobilephase
➢Columnhasaliquidorpolymerstationaryphasethatisadsorbedtothesurfaceofa
metaltube
➢Columnsare1.5-10minlengthand2-4mmininternaldiameter
258

259. ❖Componentsofgaschromatography
➢Column;
✓Packedcolumn-itsglassorstainlessstill,2or3meterlongand2-4mminternaldiameter
✓Capillarycolumn-madefromfusedquartz,10-100meterand0.1-1mmindiameter
➢Stationaryphasecontaingreaseorwaxwhichcanwithstandhightemperature
➢Mobilephase–useusuallyinertgasheliumorunreactivegasnitrogen
✓Mobilephasepassthroughmolecularsieve
✓Thesieveusedtofilterorremoveunwantedhydrocarbon,watervaporandoxygenthatmayinterfere
withthesampleduringanalysis
➢Detector
259

260. Con…
260

261. 3.Detection methods
❖MassSpectrometry(MS)
➢Analyticalmethodtomeasurethemolecularoratomicweightofsamples
➢DifferentTypesofMS
✓GC-MS-GasChromatographyMS–separatesvolatilecompoundsingascolumnand
ID’sbymass
✓LC-MS-LiquidChromatographyMS–separatesdelicatecompoundsinHPLC
columnandID’sbymass
✓MS-MS-TandemMassSpectrometry–separatescompoundfragmentsbymagneticor
electricfieldsandID’sbymassfragmentpatterns
261

262. ❖Nuclear Magnetic Resonance (NMR) Spectroscopy
➢NMRusesradio-frequency(RF)radiationandmagneticfields.
➢RFradiationisusedtostimulatenucleipresentwithinmolecules.
➢Theinformationobtainedisdisplayedasaspectrum.Thehorizontalaxisisthechemicalshift(delta,in
unitsofppm),whichisameasureofthepositionatwhichRFabsorptionoccursrelativetoaninternal
standard(tetramethylsilane,TMS).Theverticalaxisistheintensityoftheabsorption.Aswithother
spectraltechniques,compoundshavecharacteristicspectra.Morethan100metabolitesoccurinplantsat
levelshighenoughforanalysisbyNMR,soNMRspectraofmixturescontainmanypeaks.
262

263. Con…
263

264. 4.Challenges of Metabolomics
➢Metabolomicsisnotonlyconcernedwiththeidentificationand
quantificationofmetabolites,itisalsoconcernedwithrelatingmetabolite
datatobiologyandmetabolism.
➢Asaresult,metabolomicsrequiresthatwhateverchemicalinformationit
generatesmustbelinkedtobothbiochemicalcausesandphysiological
consequences.
➢Thismeansthatmetabolomicsmustcombinethetwoverydifferentfields
ofinformatics:bioinformaticsandcheminformatics.
264

265. Con…
➢Asaresult,theanalyticalsoftwareusedinmetabolomicsisfundamentallydifferent
fromanyofthesoftwareusedingenomics,proteomicsortranscriptomics.
➢Asinallfields,metabolomicsrequireelectronicallyaccessibleandsearchable
databases,allofthemrequiresoftwaretohandleorprocessdatafromtheirownhigh-
throughput
➢allofthemrequirelaboratoryinformationmanagementsystems(LIMS)tomanage
theirdata,andallrequiresoftwaretoolstopredictormodelproperties,pathways,
relationshipsandprocesses.
265

266. 5. Application of metabolomics
➢Toxicityassessment/toxicology–Urineorbloodplasmasamplescanbeusedtodetectthe
physiologicalchangescausedbytoxicinsultofachemical(ormixtureofchemicals)
➢Functionalgenomics.–Metabolomicscanbeanexcellenttoolfordeterminingthe
phenotypecausedbyageneticmanipulation,suchasgenedeletionorinsertion.
➢Moreexcitingistheprospectofpredictingthefunctionofunknowngenesby
comparisonwiththemetabolicperturbationscausedbydeletion/insertionofknown
genes.
266

267. Con…
➢PersonalizedMedicine-Individualsamplesfromthemetabolitesandtheir
concentrationscouldbeusedfordiseasediagnosisandtreatment.
➢Theresponseoftheindividualtocertainmedicationscanbedetectedbycheckingthe
metaboliteconcentrations.
➢Agriculture-Metabolomicshelpsustoimprovethegeneticallymodifiedcropsandalso
helpsustoidentifythedangersoftheconsumptionofGMcropsbymakingusgeta
snapshotoftheplantdevelopmentatdifferenttimeframes.
➢Plantmetaboliteidentificationisparticularlyimportantasitwouldhelpusin
identificationofthefunctionsoftheprimaryandsecondarymetabolites.
267

268. Cont...
➢Nutrigenomics-understandinghownutrientsaffectgenestherefore,willenablefoodsto
bedevelopedthatcanbeusedtopreventandtreatdisease.
➢Thefieldofmetabolomicscanbeappliedtonutrigenomicsindeterminingthemetabolic
fingerprintoftheorganism,
-whichwillprovidethecompletesnapshotofboththefactorsandtheireffectonthe
individualsystem.
268

269. Con…
❖Applications of Metabolomics
➢Clinical Trial Testing
➢Fermentation Monitoring & Food & Beverage Tests
➢Nutraceutical Analysis
➢Drug Phenotyping
➢Water Quality Testing
➢Petrochemical Analysis &Genetic Disease Tests
➢Nutritional Analysis
➢Clinical Blood Analysis
➢Clinical Urinalysis
➢Cholesterol Testing
➢Drug Compliance……………..
269

270. Wish you all the best
270