pcr and its applications in biotechnology

pcr and its applications in biotechnology

1. Polymerase Chain Reaction
and its Applications

2. Polymerase Chain Reaction (PCR)
•PCRisameanstoamplifyaparticularpieceofDNA
•Amplify=makingnumerouscopiesofasegmentofDNA
•PCRcanmakebillionsofcopiesofatargetsequenceofDNAin
afewhours
•PCRwasinventedbyKaryMullis(Cetuscorporation,USA)in
the1983-1984asawaytomakenumerouscopiesofDNA
fragmentsinthelaboratory
•KaryMullisawardedNobelPrizeinChemistryin1993.
•1985,SaikipublishesthefirstapplicationofPCR(beta-Globin)
•ItsapplicationsarevastandPCRisnowanintegralpartof
MolecularBiology/RDT/Biotechnology.
•1966,ThomasBrockdiscoversThermusaquaticus,a
thermostablebacteriainthehotspringsofYellowstone
NationalPark
•1985,CetusCorp.ScientistsisolateThermostableTaq
Polymerase(fromT.aquaticus),whichrevolutionizedPCR

3. DNA Replication vs. PCR
•PCRisalaboratoryversionofDNAReplicationincells–In
vitroamplificationofDNA
•Thelaboratoryversioniscommonlycalled“invitro”
sinceitoccursinatesttubewhile“invivo”signifies
occurringinalivingcell.

4. DNA Replication in Cells (in vivo)
•DNAreplicationisthecopyingofDNA
•IttypicallytakesacelljustafewhourstocopyallofitsDNA
•DNAreplicationissemi-conservative(i.e.onestrandofthe
DNAisusedasthetemplateforthegrowthofanewDNA
strand)
•Thisprocessoccurswithveryfewerrors(onaveragethereis
oneerrorper1billionnucleotidescopied)
•MorethanadozenenzymesandproteinsparticipateinDNA
replication

5. Key enzymes involved in DNA Replication
•DNA Polymerase
•Helicase
•Primase
•Topoisomerase
•Single strand binding protein
•DNA Ligase

6. DNA Replication enzymes: DNA Polymerase
•CatalyzestheelongationofDNAbyaddingnucleoside
triphosphatestothe3’endofthegrowingstrand
•Anucleotidetriphosphateisa1sugar+1base+3
phosphates
•WhenanucleosidetriphosphatejoinstheDNAstrand,
twophosphatesareremoved.
•DNApolymerasecanonlyaddnucleotidesto3’endof
growingstrand

7. Complementary Base-Pairing in DNA
•DNAisadoublehelix,madeupofnucleotides,withasugar-
phosphatebackboneontheoutsideofthehelix.
Note:anucleotideisasugar+phosphate+nitrogenousbase
•ThetwostrandsofDNAareheldtogetherbypairsofnitrogenous
basesthatareattachedtoeachotherviahydrogenbonds.
•Thenitrogenousbaseadeninewillonlypairwiththymine
•Thenitrogenousbaseguaninewillonlypairwithcytosine
•Duringreplication,oncetheDNAstrandsareseparated,DNA
polymeraseuseseachstrandasatemplatetosynthesizenewstrands
ofDNAwiththeprecise,complementaryorderofnucleotides.

8. DNA Replication enzymes: DNA Ligase
•ThetwostrandsofDNAinadoublehelixareanti-parallel(i.e.
theyareorientedinoppositedirectionswithonestrand
orientedfrom5’to3’andtheotherstrandorientedfrom3’to
5’
•5’and3’refertothenumbersassignedtothecarbonsinthe5carbon
sugar
•Giventheanti-parallelnatureofDNAandthefactthatDNA
ploymerasescanonlyaddnucleotidestothe3’end,onestrand
(referredtoastheleadingstrand)ofDNAissynthesized
continuouslyandtheotherstrand(referredtoasthelagging
strand)insynthesizedinfragments(calledOkazaki
fragments).
•OkazakifragmentsarejoinedtogetherbyDNAligase.

9. DNA Replication enzymes: Primase
•DNAPolymerasecannotinitiatethesynthesisofDNA
•RememberthatDNApolymerasecanonlyadd
nucleotidesto3’endofanalreadyexistingstrandof
DNA
•Inhumans,primaseistheenzymethatcanstartanRNAchain
fromscratchanditcreatesaprimer(ashortstretchRNAwith
anavailable3’end)thatDNApolymerasecanaddnucleotides
toduringreplication.
NotethattheRNAprimerissubsequentlyreplacedwith
DNA

10. DNA Replication enzymes
Helicase, Topoisomerase and Single-strand binding protein
•HelicaseuntwiststhetwoparallelDNAstrands
•Topoisomeraserelievesthestressofthistwisting
•Single-strandbindingproteinbindstoandstabilizesthe
unpairedDNAstrands

11. PCR: the in vitroversion of DNA Replication
ThefollowingcomponentsareneededtoperformPCRinthe
laboratory:
1)DNA(yourDNAofinterestthatcontainsthetargetsequence
youwishtocopy)
2)Aheat-stableDNAPolymerase(likeTaqPolymerase)
3)Allfournucleotidetriphosphates
4)Buffers
5)Twoshort,single-strandedDNAmoleculesthatserveas
primers
6)Thinwalledtubes
7)Thermalcycler(adevicethatcanchangetemperatures
dramaticallyinaveryshortperiodoftime)

12. PCR
TheDNA,DNApolymerase,buffer,
nucleosidetriphosphates,andprimers
areplacedinathin-walledtubeand
thenthesetubesareplacedinthePCR
thermalcycler
PCR Thermocycler

13. The three main steps of PCR
•The basis of PCR is temperature changes and the effect that these
temperature changes have on the DNA.
•In a PCR reaction, the following series of steps is repeated 20-40
times
Note: 30 cycles usually takes about 2-3 hours and amplifies the DNA
fragment of interest 1,000,000,000 fold.
Step 1: Denature DNA
At 95C for 20-30 seconds, the DNA is denatured (i.e. the two
strands are separated)
Step 2: Annealing (of Primers)
At 40C-65C, the primers anneal (or bind to) their
complementary sequences on the single strands of DNA
Step 3: Extension (of the DNA chain by DNA polymerase)
At 72C (70-80 C), DNA Polymerase extends the DNA chain by
adding nucleotides to the 3’ ends of the primers.

15. Heat-stable DNA Polymerase
•GiventhatPCRinvolvesveryhightemperatures,itis
imperativethataheat-stableDNApolymerasebeusedinthe
reaction.
•MostDNApolymeraseswoulddenature(andthusnot
functionproperly)atthehightemperaturesofPCR.
•TaqDNApolymerasewaspurifiedfromthehotsprings
bacteriumThermusaquaticusin1976
•Taqhasmaximalenzymaticactivityat75Cto80C,and
substantiallyreducedactivitiesatlowertemperatures.

16. More about Primers
•PCRprimersareshort,singlestrandedDNAmolecules(15-40
bp)
•Theyaremanufacturedcommerciallyandcanbeorderedto
matchanyDNAsequence
•Primersaresequencespecific,theywillbindtoaparticular
sequenceinagenome
•Asyoudesignprimerswithalongerlength(16→40bp),the
primersbecomemoreselective.
•DNApolymeraserequiresprimerstoinitiatereplication
•Primersbindtotheircomplementarysequenceonthetarget
DNA
–Aprimercomposedofonly3letter,ACC,forexample,would
beverylikelytoencounteritscomplementinagenome.
–Asthesizeoftheprimerisincreased,thelikelihoodof,for
example,aprimersequenceof35baselettersrepeatedly
encounteringaperfectcomplementarysectiononthetarget
DNAbecomeremote.

17. Step: 1 Denaturation of DNA
This occurs at 95 ºC mimicking the function of helicase in the cell.

18. Step 2 Annealing or Primers Binding
Primersbindtothecomplimentarysequenceonthetarget
DNA.
Primersarechosensuchthatoneiscomplimentarytotheone
strandatoneendofthetargetsequenceandthattheotheris
complimentarytotheotherstrandattheotherendofthe
targetsequence.
Forward Primer
Reverse Primer

19. Step 3 Extension or Primer Extension
DNApolymerasecatalyzestheextensionofthestrandinthe5-3
direction,startingattheprimers,attachingtheappropriate
nucleotide(A-T,C-G)
extension
extension

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

21. The DNA of interest is amplified by a power of
2 for each PCR cycle
Forexample,ifyou
subjectyourDNAof
interestto5cyclesof
PCR,youwillendup
with2
5
(or64)copiesof
DNA.
Similarly,ifyousubject
yourDNAofinterestto
40cyclesofPCR,you
willendupwith2
40
copiesofDNA!

22. Applications of PCR
•PCRisusedinanalyzingclinicalspecimensforthe
presenceofinfectiousagents,includingHIV,hepatitis,
malaria,anthrax,etc.
•PCRcanprovideinformationonapatient’sprognosis,and
predictresponseorresistancetotherapy.Manycancersare
characterizedbysmallmutationsincertaingenes,andthis
iswhatPCRisemployedtoidentify.
•PCRisusedintheanalysisofmutationsthatoccurinmany
geneticdiseases(e.g.cysticfibrosis,sicklecellanaemia,
phenylketonuria,musculardystrophy).

23. •PCR is also used in forensics laboratories and is especially
useful because only a tiny amount of original DNA is
required, for example, sufficient DNA can be obtained from a
droplet of blood or a single hair.
•PCR is an essential technique in cloning procedure which
allows generation of large amounts of pure DNA from tiny
amount of template strand and further study of a particular
gene.
•The Human Genome Project (HGP) for determining the
sequence of the 3 billion base pairs in the human genome,
relied heavily on PCR.

24. •PCRhasbeenusedtoidentifyandtoexplore
relationshipsamongspeciesinthefieldofevolutionary
biology.Inanthropology,itisalsousedtounderstandthe
ancienthumanmigrationpatterns.Inarchaeology,ithas
beenusedtospottheancienthumanrace.PCR
commonlyusedbyPaleontologiststoamplifyDNAfrom
extinctspeciesorcryopreservedfossilsofmillionsyears
andthuscanbefurtherstudiedtoelucidateon.