rDNA- Restriction Mapping, PCR, Reverse Transcription- PCR.pdf

Mapping

1. RECOMBINANT DNA
TECHNOLOGY
Unit III

2. Restriction Mapping
•Restrictionmappingisaphysicalmappingtechniquewhichisusedto
determinetherelativelocationofrestrictionsitesonaDNAfragment
togivearestrictionmap.
•Restrictionenzymesareendonucleases-oneofthemostimportanttoolsin
therDNAtechnologythatrecognizespecificsequencesonDNAandmake
specificcuts.
•Theseenzymesareproducedbybacteriatoprotectthemselvesfrom
bacteriophageattack.
•Theseareclassifiedintofourgroups:typeI,typeII,typeIIIandtypeIV.
TypeIIenzymesaremostfrequentlyusedformoleculartechniques.

3. Restriction enzymes
•TheseareDNAcuttingenzymepresentinbacteria(prokaryotes)thatrecognizesthe
specificsitesintheDNA,calledrestrictionsitesandmakeaDNAdoublestrand
breakatornearrecognitionsites.
•Aftercutting,thenewlyproducedDNAendswillhaveeitherabluntendedor
sticky(staggered)end.

4. Restriction Enzyme/Endonuclease
RestrictionEnzyme/Endonuclease“Alsoknownasmolecularscissor”
•TypeIenzymes:Theyarecomplexmultisubunitcombinationrestrictionand
modificationenzymesthatcutDNAatrandomfarfromtheirrecognition
sequences
•TypeIIenzymes:CutDNAatdefinedpositionsclosetoorwithintheir
recognitionsequences
•TypeIIIenzymes:Theycleaveoutsideoftheirrecognitionsequencesand
requiretwosuchsequencesinoppositeorientationswithinthesameDNA
moleculetoaccomplishcleavagetheyrarelygivecompletedigests
•TypeIVenzymes:Recognizemodified,typicallymethylatedDNAandcleave
atthatregion

5. Restriction Mapping
•Someenzymesrecognizefourbasesasrestrictionsitesandmakefrequentcutsand
generaterelativelysmallerfragments,whilesomeenzymesrecognizesixbasesoreight
basesandmakerarecutstogeneratelargerfragments(e.g.theNotIrecognition
sequenceisGGCGCGCC).
•WhenalargegenomicDNAisdigestedwithafrequentcuttingenzyme,millionsof
DNAfragmentsaregenerated
Example of restriction enzymes and
their corresponding restriction sits

6. Restriction Mapping
•Separationofthesefragmentsbyconventionalagarosegel
electrophoresisresultsinnodiscretebands.
•Therefore,rarecuttingenzymesareusefulforgenomemappingas
theygeneraterelativelylessDNAfragments.
•Afterdigestion,DNAfragmentsareseparatedinagarosegelusinga
specialtechniquecalledPulseFieldGelElectrophoresis(PFGE).
•Usingthistechnique,DNAfragmentsofupto10Mbpcanbe
separatedasagainstlessthan40kbpwhichcanbeseparatedusing
conventionalagarosegelelectrophoresis

7. Steps in Restriction Mapping
Preparation of DNA for restriction analysis.
Restriction digestion of DNA.
Separation of restricted DNA.
Collecting data.
Construction of restriction map

8. Restriction Mapping

9. Restriction Mapping
•Restrictionmappingofthegenomeisusefulonlyforsmallergenomessuchas
virusesandbacteria.
•RestrictiondigestionofthelargegenomesproducesmanyDNAfragments.
Whenthesefragmentsareseparatedinagarosegel,DNAfragmentscannotbe
viewedasdiscretebands.
•Anotherversionofrestrictionmappingcalledopticalmappingisusedfor
largergenomes.
•Inthismethod,DNAfragmentsarenotseparatedinagaroseafterdigestion.
•Restrictiondigestionisdonebyplacingtheisolatedchromosomeinagarose
solutiononamicroscopicslideandallowingageltoform.

10. Restriction Mapping
•Whentheagaroseformsthegel,theDNAisstretched.The
restrictionenzymeisfloodedontheagarosegelcontainingDNA
andthemixtureisincubated.
•ThentherestrictedDNAisobservedunderahighpowermicroscope
andtherelativelocationoftherestrictionsitesarevisualizedas
gaps.
•Fromthelocationofgaps,therestrictionssitesaremapped.
Saraswathy(2011). Concepts and Techniques in Genomics and Proteomics, WoodheadPublishing Limited, 77-93.

11. Application of Restriction Mapping
•Identificationofrestrictionsites
•Insertionanddeletionstudyofagene
•Insertanalysisduringcloning
•Verificationofthesizeoftheinsertduringcloning
•Mutationstudies

12. Restriction Mapping
Overview
•RestrictionmappingisamethodusedtomapanunknownsegmentofDNAbybreakingitintopiecesand
thenidentifyingthelocationsofthebreakpoints.
•Thismethodreliesupontheuseofproteinscalledrestrictionenzymes,whichcancut,ordigest,DNA
moleculesatshort,specificsequencescalledrestrictionsites
•AfteraDNAsegmenthasbeendigestedusingarestrictionenzyme,theresultingfragmentscanbe
examinedusingalaboratorymethodcalledgelelectrophoresis,whichisusedtoseparatepiecesofDNA
accordingtotheirsize.
•OnecommonmethodforconstructingarestrictionmapinvolvesdigestingtheunknownDNAsamplein
threeways.Here,twoportionsoftheDNAsampleareindividuallydigestedwithdifferentrestriction
enzymes,andathirdportionoftheDNAsampleisdoubledigestedwithbothrestrictionenzymesatthe
sametime
•Next,eachdigestionsampleisseparatedusinggelelectrophoresis,andthesizesoftheDNAfragments
arerecorded.

13. Restriction Mapping
•Thetotallengthofthefragmentsineachdigestionwillbeequal.However,becausethelengthof
eachindividualDNAfragmentdependsuponthepositionsofitsrestrictionsites,eachrestrictionsite
canbemappedaccordingtothelengthsofthefragments.
•Theinformationfromthedoubledigestionisparticularlyusefulforcorrectlymappingthesites.
•ThefinaldrawingoftheDNAsegmentthatshowsthepositionsoftherestrictionsitesiscalleda
restrictionmap
•ItispossibletodeterminethepositionofrestrictionsitesonaDNAfragmentbydigestingitwith
variousrestrictionenzymes,singlyandincombination,andanalyzingthefragmentsizesobtained.
•RestrictionmappingusedtobeakeyprocedureforcharacterizingaclonedfragmentofDNA,
butthisisnowmoreeasilydonebyDNAsequencing.
•Nevertheless,analysisofrestrictionsites(orfragmentsizes)isstilluseful,forexamplein
comparingthechromosomalorganizationofdifferentstrains.

14. PCR
•PCRisarevolutionarymethoddevelopedbyKaryMullisinthe1980s.
•PCRisatechniquethattakesaspecificsequenceofDNAofsmallamountsand
amplifiesittobeusedforfurthertesting
•PCRisbasedonusingtheabilityofDNApolymerasetosynthesizenewstrandof
DNAcomplementarytotheofferedtemplatestrand.
•BecauseDNApolymerasecanaddanucleotideonlyontoapreexisting3'-OH
group,itneedsaprimertowhichitcanaddthefirstnucleotide.
•Thisrequirementmakesitpossibletodelineateaspecificregionoftemplate
sequencethattheresearcherwantstoamplify.AttheendofthePCRreaction,the
specificsequencewillbeaccumulatedinbillionsofcopies(amplicons)
The targets in PCR are the sequences of DNA on each end of region of
interest, which can be a complete gene or a small sequence

15. Principles of PCR
•ThewholeprocessofPCRinvolvesthreemainevents,Denaturation,Annealing
andElongation.
•ADNAfragmentofinterestisusedasatemplateandapairofprimerswhichare
shortoligonucleotidescomplimentarytothebothstrandsofthetemplateDNA.
•ThepurposeofprimeristoinitiatetheDNAsynthesisinthedirectionof5‟to
3‟.
•ThenumberofamplifiedDNAortheampliconsincreasesexponentiallyper
cyclethusonemoleculeofDNAgivesriseto2,4,8,16andsoforth.
•ThiscontinuousdoublingiscarriedoutbyaspecificenzymecalledDNA
polymerasewhichsitsattheunfinisheddoublestrandedDNAcreatedbytemplate
DNAandprimer.

16. Principles of PCR
•ForfurtherextensionoftheDNA,thepolymeraseenzymerequiresupplyofother
DNA-buildingblockssuchasthenucleotidesconsistingoffourbasesAdenine(A),
Thymine(T),Cytosine(C)andGuanine(G).
•Thetemplate,primer,polymeraseandfourbasesarethemaincomponentsfor
polymerasechainreaction.
Note*
•Initialdenaturation:HeatingthePCRmixtureat94°Cto96°Cfor10mintoensure
completedenaturationoftemplateDNA
•Afterthecyclesarecompleted,thereactionisheldat70-74°Cforseveralminutes
toallowfinalextensionoftheremainingDNAtobefullyextended.

17. Components of PCR
DNAtemplate:
•thesampleDNAthatcontainsthetargetsequence.Atthebeginningofthereaction,high
temperatureisappliedtotheoriginaldouble-strandedDNAmoleculetoseparatethe
strandsfromeachother.
DNApolymerase:
•atypeofenzymethatsynthesizesnewstrandsofDNAcomplementarytothetarget
sequence.
•ThefirstandmostcommonlyusedoftheseenzymesisTaqDNApolymerase(from
Thermisaquaticus),whereasPfuDNApolymerase(fromPyrococcusfuriosus)isused
widelybecauseofitshigherfidelitywhencopyingDNA.
•Althoughtheseenzymesaresubtlydifferent,theybothhavetwocapabilitiesthatmake
themsuitableforPCR:1)theycangeneratenewstrandsofDNAusingaDNA
templateandprimers,and2)theyareheatresistant.

18. Components of PCR
Primers
•Shortpiecesofsingle-strandedDNAthatarecomplementarytothetargetsequence.
•ThepolymerasebeginssynthesizingnewDNAfromtheendoftheprimer.
Nucleotides(dNTPsordeoxynucleotidetriphosphates)
•SingleunitsofthebasesA,T,G,andC,whichareessentially"buildingblocks"fornewDNA
strands.
Mg++ions-cofactoroftheenzyme
Buffersolution
•MaintainspHandionicstrengthofthereactionsolutionsuitablefortheactivityoftheenzyme
RT-PCR
•ReverseTranscriptionPCRisPCRprecededwithconversionofsampleRNAintocDNAwith
enzymereversetranscriptase
https://www.ncbi.nlm.nih.gov/probe/docs/techpcr/

19. Primer Designing & Criteria
•PrimerLength–18-24basesistheideallength
•Primermeltingtemperature–52-58°C
•Primerannealingtemperature
•GCContent–40-60%
•GCClamp
•Primersecondarystructures
–Hairpins
–Dimers

20. PCR process

21. PCR process

22. PCR Process
Step1:Denaturation
•AsinDNAreplication,thetwostrandsintheDNAdouble
helixneedtobeseparated.
•Theseparationhappensbyraisingthetemperatureofthemixture
(94-95°C),causingthehydrogenbondsbetweenthecomplementary
DNAstrandstobreak.Thisprocessiscalleddenaturation.

23. PCR Process
Step2:Annealing
•PrimersbindtothetargetDNA sequencesand
initiatepolymerization.
•Thiscanonlyoccuroncethetemperatureofthesolutionhasbeen
lowered(55-65°C).Oneprimerbindstoeachstrand.

24. PCR Process
Step3:Extension
•NewstrandsofDNAaremadeusingtheoriginalstrandsastemplatesat
72°C
•ADNApolymeraseenzymejoinsfreeDNAnucleotidestogether.
•ThisenzymeisoftenTaqpolymerase,anenzymeoriginallyisolatedfrom
athermophilicbacteriacalledThermusaquaticus.
•Theorderinwhichthefreenucleotidesareaddedisdeterminedbythe
sequenceofnucleotidesintheoriginal(template)DNAstrand.
•TheresultofonecycleofPCRistwodouble-strandedsequencesoftarget
DNA,eachcontainingonenewlymadestrandandoneoriginalstrand.

25. PCR Process
•Thecycleisrepeatedmanytimes(usually20–30)asmostprocesses
usingPCRneedlargequantitiesofDNA.
•Itonlytakes2–3hourstogetabillionorsocopies.

26. Applications of PCR
Basic Research
•Mutation screening
•Drug discovery
•Classification of organisms
•Genotyping
•Molecular Archaeology
•Molecular Epidemiology
•Bioinformatics
•Genomic cloning
•Site-directed mutagenesis
•Gene expression studies
Applied Research
•Genomic matching
•Detection of pathogens
•Pre –natal diagnosis
•DNA fingerprinting
•Gene therapy

27. Application of PCR
PCRinhumanmedicine
•PCRtechnologyhasbecomeanessentialresearchanddiagnostic
toolforimprovinghumanhealthandqualityoflife.
•Itallowsthedetectionofinfectiousorganismsjustfromonecellby
amplifyingspecificregionofthegeneticmaterial.
•SomeimportantareasinmedicalresearchwherePCRtechnologyis
employedincludethefollowing

28. Application of PCR
PCRininfectiousdisease
•PCRtechnologyhasbecomethebasisforabroadspectrumofclinical
diagnostictestsforvariousinfectiousagents,includingvirusesandbacteria
•Besidesdetectingthepresenceofpathogens,PCRallowsustoquantifythe
amountofpathogenspresentinpatient’sbloodandthishelpstomonitorthe
progressionofinfectionorresponsetodrugtreatment.
•PCRhasenabledthedevelopmentofdiagnostictestsformanydiseases
suchas,HIV-1,HepatitisBandCviruses,HumanPapillomavirus,
Chlamydiatrachomatis,Neisseriagonorrhoeae,Cytomegalovirus,
Mycobacteriumtuberculosis.

29. Application of PCR
PCRandgenetictesting:
•PCRtechnologyhasrecentlybecomeapowerfultooltodetect
diseaseassociatedgenetopredictthepresenceofheartdiseaseand
cancers.
•Knowledgeofdiseaseassociatedgeneinthepersonpredisposedto
thesedisordershaveachancetocontroltheproblemmuchin
advance.
.

30. Application of PCR
PCRinplantscience:
•TherearevariousfieldsinplantsciencewhichrequirestheuseofPCR
technologyforitsaccomplishment
Plantspeciesidentification:
•PCRtechniquehasalsobeenemployedinidentificationofplantspeciesusing
speciesandgroup-specificprimerstargetingchloroplastDNA.
•Theseassaysallowedidentificationofplantsbasedonsize-specificamplicons.
plantsbelongingtothesamefamilyhascloseprimer-bindingsitesandhence
sameampliconssizewhileplantsbelongingtodifferentspeciesandgroups
havedifferentprimer-bindingsitesandhencewillresultindifferentamplicons
size.

31. Application of PCR
PCRintissueculture:
•ItisusedinanalysisofDNAandspecificgenesinplantcellsat
differentstagesofre-generationduringinvitroculturealongwith
RAPD(randomamplificationofpolymorphicDNA)technology.
•Thelevelofpolymorphisminregeneratedplantscouldberevealed
bythesedualtechniques.

32. Application of PCR
PCRinveterinaryparasitology:
•Owingtoitsrapidityandsensitivityascomparedtoantibody-baseddiagnosis,PCRmet
itsusesinalmostallaspectsofbiologicalworkincludingveterinaryclinicaldiagnosis.
•SomeexamplesofPCRapplicationsinveterinaryparasitology:
Aujeszky’sdisease(pseudorabis)virusofpigs:
•Thisviruscausesabortionandmortalityinpiglets.
•Thisdiseasehasalatentperiodwherethereisnosymptomofinfectionmakingit
difficulttoeradicatethediseasecompletely.
•Forthisreason,PCRisconsideredtobeappropriatetoolfordetectinglatentcasesof
Aujeszky‟sdisease.

33. Application of PCR
Bovineleukemiavirus(BLV):
•Thisviruscausesenzooticbovineleukosis.PCRassayfordetectionofBLVwas
developedin1991.
Bovineviraldiarrhoeavirus(BVDV):
•Thisvirusisnotonlyfataltocattlebutalsocausescontaminationincalfserumusedin
cellcultureworkthusleadingtocontaminationofvaccinesandpharmaceuticalproducts.
•Besidestheaboveexamples,PCRhasbeenusedinroutinediagnosisofveterinaryvirus
suchasPorcineparvo,Bovinepapillomatype1and2,Avianpolyoma,Chickenanemia,
Duckhepatitis,Africanswinefever,Channelcatfish,Equineherpestype1and4,Feline
herpes,Alcelaphineherpestype1etc.

34. Application of PCR
PCRinForensicapplications:
•ThemostcommonuseofPCRinforensicapplications
includes:
Criminalinvestigation:
•Asampleofblood,hairrootortissueleftinthecrime
scenecanbeusedtoidentifyapersonusingPCRby
comparingtheDNAofthecrimescenewiththatof
suspectorwithDNAdatabaseofearlierconvicts.
•Evidencefromdecades-oldcrimescanbetested,
confirmingordefendingthepeopleoriginally
convicted.

35. Application of PCR
Parentaltesting:
•PCRtechnologyisalsousedinfindingthebiological
parentsofadoptedorkidnappedchildwheretheDNAof
achildismatchedwithcloserelatives
•Theactualbiologicalfatherofanewborncanalsobe
ruledout.
•Inparentaltesting,shorttandemrepeats(STR)areused
asmarkerswhereeachperson’sDNAcopiescontaintwo
copiesofthesemarkersoneeachfromfatherandmother.
•Thesemarkersdifferinlengthandsometimessequence.

36. Application of PCR
PCRinresearchapplications:
•Biologicalresearchrequiresmolecularbiologytechniquesasitsstarting
materialandsoforthwhichcannotbeaccomplishedwithouttheuseofPCR.
DNAcloning:
•PCRhelpstoamplifyspecificDNAfromagenomeandtheamplifiedDNAcan
beinsertedintoavectorfortransformationandexpression.
•TheseinsertscanfurtherbeconfirmedbyPCRmethod.
DNAsequencing:
•PCRassiststhetaskofDNAsequencingfrompatientswithgeneticdisease
mutation.

37. Application of PCR
Sequence-taggedsites:
•ThisisaprocesswherePCRisusedasanindicatorifaparticularsegment
ofageneorgenomeispresentinaparticularclone.
•Thisapplicationisvitalinmappingthecosmidclonestobesequencedby
theHumanGenomeProject.
Phylogenyanalysis:
•Thephylogenyoforganismslikeplants,animalsandotherlowerorganisms
canbetracedoutbyDNAanalysis.
•Theoriginofunknownsamplesliketherecoveredbonesofearlymencan
alsoberulesout.

38. Reverse Transcription PCR (RT-
PCR
•ReversetranscriptionPCR(RT-PCR)usesmRNAratherthanDNAasthestarting
template.
•First,theenzymereversetranscriptaseusesthemRNAtemplatetoproducea
complementarysingle-strandedDNAstrandcalledcDNAinaprocessknownasreverse
transcription.
•Next,DNApolymeraseisusedtoconvertthesingle-strandedcDNAintodouble-
strandedDNA.
•TheseDNAmoleculescannowbeusedastemplatesforaPCRreactionasdescribed
above.
•ThevalueofRT-PCRisthatitcanbeusedtodetermineifanmRNAspeciesispresent
inasampleortocloneacDNAsequenceforasubsequentexperiment.
Guide to Research Techniques in Neuroscience. http://dx.doi.org/10.1016/B978-0-12-800511-8.00010-1

39. Reverse Transcription PCR (RT-
PCR
RT-PCRhasrisentobecomethebenchmarktechnologyforthedetection
and/orcomparisonofRNAlevelsforseveralreasons:
•itdoesnotrequirepostPCRprocessing,
•awiderange(>10
7
-fold)ofRNAabundancecanbemeasured,and
•itprovidesinsightintobothqualitativeandquantitativedata.
Duetoitssimplicity,specificityandsensitivity,RT-PCRisusedinawide
rangeofapplicationsfromexperimentsassimpleasquantificationofyeast
cellsinwinetomorecomplexusesasdiagnostictoolsfordetectinginfectious
agentssuchastheavianfluvirusandSARS-CoV-2.
Guide to Research Techniques in Neuroscience. http://dx.doi.org/10.1016/B978-0-12-800511-8.00010-1

40. Reverse Transcription PCR (RT-
PCR) Principle
•InRT-PCR,theRNAtemplateisfirstconvertedinto
acomplementaryDNA(cDNA)usingareversetranscriptase(RT).
•ThecDNAisthenusedasatemplateforexponentialamplification
usingPCR.
•TheuseofRT-PCRforthedetectionofRNAtranscripthas
revolutionizedthestudyofgeneexpressioninthefollowing
importantways:
–Madeittheoreticallypossibletodetectthetranscriptsofpracticallyany
gene
[
–Enabledsampleamplificationandeliminatedtheneedforabundantstarting
materialrequiredwhenusingnorthernblotanalysis
–ProvidedtoleranceforRNAdegradationaslongastheRNAspanningthe
primerisintact
Guide to Research Techniques in Neuroscience. http://dx.doi.org/10.1016/B978-0-12-800511-8.00010-1

41. Principles
One-stepRT-PCRvstwo-stepRT-PCR
•ThequantificationofmRNAusingRT-PCRcanbeachievedaseitheraone-steporatwo-stepreaction.
•Thedifferencebetweenthetwoapproachesliesinthenumberoftubesusedwhenperformingthe
procedure.
•Thetwo-stepreactionrequiresthatthereversetranscriptasereactionandPCRamplificationbe
performedinseparatetubes.
•Thedisadvantageofthetwo-stepapproachissusceptibilitytocontaminationduetomorefrequent
samplehandling.
•Ontheotherhand,theentirereactionfromcDNAsynthesistoPCRamplificationoccursinasingle
tubeintheone-stepapproach.
•Theone-stepapproachisthoughttominimizeexperimentalvariationbycontainingallofthe
enzymaticreactionsinasingleenvironment.IteliminatesthestepsofpipettingcDNAproduct,whichis
labor-intensiveandpronetocontamination,toPCRreaction.

42. Principles
•Thefurtheruseofinhibitor-tolerantpolymerases,polymeraseenhancerswithanoptimizedone-step
RT-PCRcondition,supportsthereversetranscriptionoftheRNAfromunpurifiedorcrudesamples,
suchaswholebloodandserum.
•However,thestartingRNAtemplatesarepronetodegradationintheone-stepapproach,andtheuse
ofthisapproachisnotrecommendedwhenrepeatedassaysfromthesamesampleisrequired.
•Additionally,theone-stepapproachisreportedtobelessaccuratecomparedtothetwo-step
approach.
•ItisalsothepreferredmethodofanalysiswhenusingDNAbindingdyessuchasSYBRGreen
sincetheeliminationofprimer-dimerscanbeachievedthroughasimplechangeinthemelting
temperature
•Nevertheless,theone-stepapproachisarelativelyconvenientsolutionfortherapiddetection
oftargetRNAdirectlyinbiosensing.

43. Principles
End-pointRT-PCRvsreal-timeRT-PCR
•QuantificationofRT-PCRproductscanlargelybedividedintotwocategories:
–end-point
–real-time
•Theuseofend-pointRT-PCRispreferredformeasuringgeneexpression
changesinsmallnumberofsamples
•Thereal-timeRT-PCRhasbecomethegoldstandardmethodforvalidating
quantitativeresultsobtainedfromarrayanalysesorgeneexpressionchangeson
aglobalscale.

44. Principles
End-pointRT-PCR
•Themeasurementapproachesofend-pointRT-PCRrequiresthedetection
ofgeneexpressionlevelsbytheuseoffluorescentdyeslikeethidium
bromide,P32labelingofPCRproductsusingphosphorimageror
byscintillationcounting.
•End-pointRT-PCRiscommonlyachievedusingthreedifferentmethods:
relative,
competitive
comparative

45. Application
•Theexponentialamplificationviareversetranscriptionpolymerasechain
reactionprovidesforahighlysensitivetechniqueinwhichaverylowcopy
numberofRNAmoleculescanbedetected.
•RT-PCRiswidelyusedinthediagnosisofgeneticdiseasesand,
semiquantitatively,inthedeterminationoftheabundanceofspecific
differentRNAmoleculeswithinacellortissueasameasureofgene
expression

46. Application
Researchmethods
•RT-PCRiscommonlyusedinresearchmethodstomeasuregeneexpression.
•Forexample,Linetal.usedqRT-PCRtomeasureexpressionofGalgenesinyeastcells.
•First,Linetal.engineeredamutationofaproteinsuspectedtoparticipateinthe
regulationofGalgenes.
•ThismutationwashypothesizedtoselectivelyabolishGalexpression.Toconfirmthis,
geneexpressionlevelsofyeastcellscontainingthismutationwereanalyzedusingqRT-
PCR.
•Theresearcherswereabletoconclusivelydeterminethatthemutationofthisregulatory
proteinreducedGalexpression.
•NorthernblotnalysisisusedtostudytheRNA'sgeneexpressionfurther.

47. Application
Geneinsertion
•RT-PCRcanalsobeveryusefulintheinsertionofeukaryoticgenes
intoprokaryotes.
•Becausemosteukaryoticgenescontainintrons,whicharepresentinthe
genomebutnotinthematuremRNA,thecDNAgeneratedfromaRT-PCR
reactionistheexact(withoutregardtotheerror-pronenatureofreverse
transcriptases)DNAsequencethatwouldbedirectlytranslated
intoproteinaftertranscription.
•Whenthesegenesareexpressedinprokaryoticcellsforthesakeof
proteinproductionorpurification,theRNAproduceddirectlyfrom
transcriptionneednotundergosplicingasthetranscriptcontains
onlyexons.
•(Prokaryotes,suchasE.coli,lackthemRNAsplicingmechanismof
eukaryotes).

48. Application
Geneticdiseasediagnosis
•RT-PCRcanbeusedtodiagnosegeneticdiseasesuchasLesch–Nyhansyndrome.
•ThisgeneticdiseaseiscausedbyamalfunctionintheHPRT1gene,whichclinically
leadstothefataluricacidurinarystoneandsymptomssimilartogout.
•AnalyzingapregnantmotherandafetusformRNAexpressionlevelsofHPRT1will
revealifthemotherisacarrierandifthefetuswilllikelytodevelopLesch–Nyhan
syndrome.
Cancerdetection
•ScientistsareworkingonwaystouseRT-PCRincancerdetectiontohelp
improveprognosis,andmonitorresponsetotherapy.Circulatingtumorcellsproduce
uniquemRNAtranscriptsdependingonthetypeofcancer.Thegoalistodetermine
whichmRNAtranscriptsserveasthebestbiomarkersforaparticularcancercelltype
andthenanalyzeitsexpressionlevelswithRT-PCR.
[43]
•RT-PCRiscommonlyusedinstudyingthegenomesofviruseswhosegenomesare
composedofRNA,suchasInfluenzavirusA,retroviruseslikeHIVandSARS-CoV-2.
[44]
•Challe