APPLICATION OF PCR(SEMINAR).ppt

APPLICATION OF PCR

1. A SEMINAR
ON
APPLICATION OF PCR IN PLANT GENOME ANALYSIS
Submitted by-Miss. TruptiA. Raut
Roll No.: 15 (M. pharmacyFirstYear)
[Pharmacognosy & Phytochemistry]
Guided by :Archana Mohod Mam
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GOVERNMENT COLLEGE OF PHARMACY, AMRAVATI

2. CONTENTS OF SLIDES
Sr. no.Contents Slideno.
1 Introduction 3
2 Short history of PCR 5
3 DNAReplication Vs PCR 6
4 KeyEnzymes Involved in DNA Replication8
5 Three MainSteps of PCR 16
6 Primers 24
7 Applicationof PCR –Genetic Diseases 25
8 Applicationof PCR in Crop Improvement29
9 Application of PCR in short 30
10 Reference 31
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3. INTRODUCTION
•WhatisPCR?
•PCRisatechniquethattakesspecificsequenceofDNAof
smallamountandamplifiesittobeusedforfurther
testing.
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4. POLYMERASE CHAIN REACTIONS (PCR)
•PCRisameanstoamplifyaparticularpieceofDNA.
•Amplify=makingnumerouscopiesofasegmentofDNA.
•PCRcanmakebillionsofcopiesofatargetsequenceofDNAina
fewhours.
•PCRwasinventedbyKaryMullis(Cetuscorporation,USA)inthe
1983-1984asawaytomakenumerouscopiesofDNAfragmentsin
thelaboratory.
•KaryMullisawardedNobelPrizeinchemistryin1993.
•1985,SaikipublishesthefirstapplicationofPCR(beta–Globin)
•ItsapplicationarevastandPCRisnowanintegralpartofMolecular
Biology/Biotechnology
•1976,ThomasBrockdiscoversThermusaquaticus,athermostable
bacteriainthehotspringsofYellowstoneNationalPark.
•1985,CetusCorp.ScientistsisolateThermostableTaqPolymerase
(fromT.aquaticus),whichrevolutionizedPCR.
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5. SHORT HISTORY OF PCR
•1983:Dr.KaryMullisdevelopedPCR.
•1985:FirstpublicationofPCRbyCetusCorporationappears
inscience.
•1976:PurifiedTaqpolymeraseisfirstusedinPCR.
•1988:PerkinElmerintroducestheautomatedthermalcycler.
•1989:SciencedeclaresTaqpolymerase“moleculeofthe
year.”
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6. DNA REPLICATION Vs PCR
•PCR is a laboratory version of DNA Replication in cells –In
vitro amplification of DNA.
•The laboratory version is commonly called “in vitro” since it
occurs in a test tube while “in vivo” signifies occurring in a
living cell.
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7. DNA REPLICATION IN CELLS (in vivo)
•DNA replication is the copying of DNA.
•It typically takes a cell just a few hours to copy all of its DNA.
•DNA replication is semi-conservative (i.e. one strand of the DNA is
used as the template for the growth of a new DNA strand)
•This process occurs with very few errors ( on average there is one
error per 1 billion nucleotides copied)
•More than a dozen enzymes and proteins participate in DNA
replication.
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8. KEY ENZYMES INVOLVED IN DNA
REPLICATION
•DNA Polymerase
•Helicase
•Primase
•Topoisomerase
•Single strand binding protein
•DNA Ligase
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9. DNA REPLICATION ENZYMES: DNA POLYMERASE
CatalyzestheelongationofDNAbyaddingnucleoside
triphosphatetothe3’endofthegrowingstrand.
Anucleotidetriphosphateisa1sugar+1base+3phosphates
WhenanucleosidetriphosphatejoinstheDNAstrand,two
phosphatesareremoved.
DNApolymerasecanonlyaddnucleotidesto3’endof
growingstrand.
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10. COMPLEMENTARY BASE –PAIRING IN DNA
•DNAisadoublehelix,madeupofnucleotides,withasugar–
phosphatebackboneontheoutsideofthehelix.
Note:anucleotideisasugar+phosphate+nitrogenousbase.
•ThetwostrandsofDNAareheldtogetherbypairsofnitrogenous
basesthatareattachedtoeachotherviahydrogenbonds.
•Thenitrogenousbaseadeninewillonlypairwiththymine.
•Duringreplication,oncetheDNAstrandsareseparated,DNA
polymeraseuseseachstrandasatemplatetosynthesizenew
strandsofDNAwiththeprecise,complementaryorderof
nucleotides.
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11. DNA REPLICATION ENZYMES: DNA LIGASE
•The two strands of DNA in a double helix are anti-parallel (i.e.
they are oriented in opposite directions with one strand oriented
from 5’ to 3’ and the other strand oriented from 3’to 5’
•Note : 5’ to 3’ refer to the numbers assigned to the carbons in the 5
carbon sugar.
•Given the anti-parallel nature of DNA and the fact that DNA
polymerases can only add nucleotides to the 3’ end, one strand
(referred to as the leading strand) of DNA is synthesized
continuously and the other strand (referred to as the lagging strand)
in synthesized in fragments (called Okazaki fragments).
•Okazaki fragments are joined together by DNA ligase.
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12. DNA REPLICATION ENZYMES: PRIMASE
DNAPolymerasecannotinitiatethesynthesisofDNA.
RememberthatDNApolymerasecanonlyaddnucleotidesto
3’endofanalreadyexistingstrandofDNA.
Inhumans,primaseistheenzymethatcanstartanRNAchain
fromscratchanditcreatesaprimer(ashortstretchRNAwith
anavailable3’end)thatDNApolymerasecanaddnucleotides
toduringreplication.
NotethattheRNAprimerissubsequentlyreplacedwithDNA.
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13. DNA REPLICATION ENZYMES
(Helicase, Topoisimerase and Single -strand binding
protein)
•Helicase untwists the two parallel DNA strands.
•Topoisomerase relieves the stress of this twisting.
•Single-strand binding protein binds to and stabilizes the
unpaired DNA strands.
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14. PCR : The in vitro version of DNA Replication
•The following components are needed to perform PCR in the
laboratory:
1) DNA (your DNA of interest that contains the target sequence you
wish to copy)
2) A heat-stable DNA polymerase (like TaqPolymerase)
3) All four nucleotide triphosphates
4) Buffers
5) Two short, single stranded DNA molecules that serve as primers
6) Thin walled tubes
7) Thermal cycler (a device that can change temperatures dramatically
in a very short period of time).
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15. PCR
•The DNA , DNA polymerase,
buffer, nucleoside triphosphates,
and primers placed in a thin –
walled tube and then these tubes
are placed in the PCR thermal
cycler.
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16. THE THREE MAIN STEPS OF PCR
•ThebasisofPCRistemperaturechangesandtheeffectthatthese
temperaturechangeshaveontheDNA.
•InaPCRreaction,thefollowingseriesofstepsisrepeated
20-40times.
Note–30cyclesusuallytakesabout2-3hoursandamplifiestheDNA
fragmentofinterest1,000,000,000fold.
Step1:DenatureDNA
At95
0
C,theDNAisdenatured(i.e.thetwostrandsareseparated)
Step2:Annealing(ofPrimers)
At40
0
C-65
0
C,theprimersanneal(orbindto)theircomplementary
sequencesonthesinglestrandsofDNA.
Step3:Extension(oftheDNAchainbyDNApolymerase)
At72
0
C,DNAPolymeraseextendstheDNAchainbyadding
nucleotidestothe3’endsoftheprimers.
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17. 17
HEATSTABLEDNAPOLYMERASE
•GiventhatPCRinvolvesveryhightemperaturesitis
imperativethataheat–stableDNApolymerasebeusedin
thereaction.
•MostDNApolymeraseswoulddenature(andthusnot
functionproperly)atthehightemperaturesofPCR.
•TaqDNApolymerasewaspurifiedfromthehotsprings
bacteriumThermusaquaticusin1976.
•Taqhasmaximalenzymaticactivityat75
0
Cto80
0
C,and
substantiallyreducedactivitiesatlowertemperatures.
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18. Step 1 : Denaturation of DNA
This occurs at 95
0
C mimicking the function of helicasein the
cell.
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19. Step 2: Annealing or Primers Binding
ReversePrimer
ForwardPrimer
Primersbindtothecomplimentarysequenceonthetarget
DNA.
Primersarechosensuchthatoneiscomplimentarytotheone
strandatoneendofthetargetsequenceandthattheotheris
complimentarytotheotherstrandattheotherendofthetarget
sequence.
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20. Step 3: Extension or Primer Extension
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extension
extension
DNApolymerasecatalyzestheextensionofthestrandinthe5-
3direction,startingattheprimers,attachingtheappropriate
nucleotide(A-T,C-G)

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•The next cycle will begin by denaturing the new DNA
strands formed in the previous cycle.

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ThesizeoftheDNAFragmentProducedinPCRisDependent
onthePrimers.
•ThePCRreactionwillamplifytheDNAsectionbetweenthe
twoprimers.
•IftheDNAsequenceisknown,primerscanbedevelopedto
amplifyanypieceofanorganism’sDNA.

23. PCR has become a very powerful tool in molecular
biology
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•Onecanamplifyfragmentsofinterestinanorganism’sDNA
bychoosingtherightprimers.
•Onecanusetheselectivityoftheprimerstoidentifythe
likelihoodofanindividualcarryingaparticularalleleofa
gene.
•Onecanstartwithasinglespermcellorstrandofhairand
amplifytheDNAsufficientlytoallowforDNAanalysisanda
distinctivebandonanagarosegel.

24. More about Primers
•PCRprimersareshort,singlestrandedDNAmolecules(15-40bp)
•Theyaremanufacturedcommerciallyandcanbeorderedtomatch
anyDNAsequence.
•Primersaresequencespecific,theywillbindtoaparticular
sequenceinagenome.
•Asyoudesignprimerswithalonerlength(16-40bp),theprimers
becomemoreselective.
•DNApolymeraserequiresprimerstoinitiatereplication.
•PrimersbindtotheircomplementarysequenceonthetargetDNA.
•Aprimercomposedofonly3letter,ACC,forexample,wouldbe
verylikelytoencounteritscomplimentinagenome.
•Asthesizeoftheprimerisincreased,thelikelihoodof,for
example,aprimersequenceof35baselettersrepeatedly
encounteringaperfectcomplementarysectiononthetargetDNA
becomeremote.
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25. APPLICATION OF PCR: GENETIC DISEASE
•Primers can be created that will only bind and amplify certain
alleles of genes or mutations of genes.
•This is the basis of genetic counselling and PCR is used as part
of the diagnostic tests for genetic diseases.
•Some diseases that can be diagnosed with the help of PCR :
Huntington’s disease
Cystic fibrosis
Human immunodeficiency virus.
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26. 26
Huntington’s Disease (HD)
HDisageneticdisordercharacterizedbyabnormalbody
movementsandreducedmentalabilities.
HDiscausedbyamutationintheHuntingtin(HD)gene
Innon-HDindividuals,theHDgeneis“expanded”
Innon-HDindividuals,theHDgenehasapatterncalled
trinucleotiderepeatswith“CAG”occurringinrepetitionless
than30times.
InHDindividuals,the“CAG”trinucleotiderepeatoccurs
morethan36timesintheHDgene.
TheDNAisamplifiedviaPCRandsequenced(atechnique
bywhichtheexactnucleotidesequenceisdetermined)and
thenumberoftrinucleotiderepeatsisthencounted.
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27. Cystic Fibrosis (CF)
CFisageneticdiseasecharacterizedbyseverebreathing
difficultiesandapredispositiontoinfections.
CFiscausedbymutationsinthecysticfibrosistransmembrane
conductanceregulator(CTFR)gene.
Innon-CFindividuals,theCTFRgenecodesforaproteinthat
isachlorideionchannelandisinvolvedintheproductionof
sweat,digestivejuicesandmucus.
InCFindividuals,mutationsintheCTFRgeneleadstothick
mucoussecretionsinthelungsandsubsequentpersistent
bacterialinfections.
ThepresenceofCTFRmutationsinaindividualcanbe
detectedbyperformingPCRandsequencingonthat
individual’sDNA.
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28. APPLICATION OF PCR
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29. Application of PCR technology in crop improvement
1.To increase the number of very few number of DNA template.
2.Isolation of a orthologous gene sequence by using degenerate
primers designed from closely related plant species gene sequence
alignments.
3.Amplification of gene from cDNA
4.Synthesis of complementary DNA (cDNA) from RNA isolated
from the crop using modified process of PCR called Reverse-
transcriptase PCR.
5.Identification of genetically modified crops for the presence of
transgene using PCR.
6.Screening and differentiation of diseased with viral pathogen and
healthy plants using viral gene specific primers.
7.PCR is used for DNA sequencing to determine unknown PCR-
amplified sequences, which helps in gene discovery.
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30. APPLICATION OF PCR IN SHORT
1.Direct sequencing of amplified DNA
2.Engineering of new DNA sequences
3.Site –directed mutagenesis
4.Detection of gene expression
5.Amplification of specific sequences from cDNA and genome
libraries.
6.Identification of transgenics
7.Plant genetic transformation and detection of transgenics at the
tissue and whole plant levels.
8.Determination of changes in a particular gene sequence resulting
from tissue culture (e.g. somaclonal variation)
9.A modification of PCR technology known as “inverse PCR” has
been used to determine the T DNA copy number in transgenic
plants generated by Agrobacterium –mediated transformation.
10.While the expression of foreign DNA in transformation
experiments can be determined by RTPCR.
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31. REFERENCE
1.Dr.MdRageeb,MdUsman,SanjayA.Nagdevet.al,“ATextbook
ofMedicinalPlantBiotechnology”,PeeVeePublication,pgno.
126,127.
2.https://www.slideshare.net/RaviKantAgrawal/pcr-and-its-
applications-101643128
3.https://starfishmedical.com/assets/pcr-components_med.jpeg
(diagramslideno.03)
4.https://socialsci.libretexts.org/Courses/College_of_the_Canyons/A
nthro_101%3A_Physical_Anthropology/03%3A_Cell_biology/3.0
3%3A_Basics_of_DNA_Replication(diagramslideno.10)
5.https://commons.m.wikimedia.org/wiki/File:PCR_machine.jpg
6.https://www.researchgate.net/publication/271079195_Polymerase_
Chain_Reaction_A_Robust_Technology_for_Crop_Improvement
7.https://www.slideshare.net/KarthiKumar7/pcr-types-and-
applications
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