Pcr

this section helps students how to prepare master mix solution and how to pcr. specially life life science fields such as biotechnology, biology, and medical laboratory

1. University of Gondar
Institute of Biotechnology
Techniques in Biotechnology (Biot.602)
Lecture 5
Polymerase Chain Reaction (PCR)

2. DNA Replication vs. PCR
•PCRisalaboratoryversionofDNAReplication.
•laboratoryversioniscommonlycalled“invitro”sinceit
occursinatesttubewhile“invivo”signifiesoccurringina
livingcell.
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in vitro in vivo

3. DNA Replication enzymes:
•DNAPolymerase-buildsDNAstrand
•DNALigase-joinsDNAstrandtogether
•Primase-shortRNAsequencethatservesasastartingpointfor
DNAsynthesis
•HelicaseuntwiststhetwoparallelDNAstrands
•Topoisomeraserelievesthestressofthistwisting
•Single-strandbindingproteinbindstoandstabilizesthe
unpairedDNAstrands.
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4. Con…
•DNAReplicationoccurswithveryfewerrors(onaverage
thereisoneerrorper1billionnucleotidescopied).
•IttypicallytakesacelljustafewhourstocopyallofitsDNA
•DNAreplicationissemi-conservative(i.e.onestrandofthe
DNAisusedasthetemplateforthegrowthofanewDNA
strand)
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5. Polymerase Chain Reaction (PCR)
•Polymerase: DNA polymerase
–DNA polymerase duplicates DNA
–Before a cell divides, its DNA must be
duplicated
–Chain Reaction: The product of a reaction is
used to amplify the same reaction
–Results in rapid increase in the product
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6. PCR Con’t
Discovery
•PCR was discovered by KaryMullis
–On a long motorcycle drive
–Mentally visualized the process
•Nobel Prize in Chemistry
–1993
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7. DNA polymerase
•DuplicatesDNA
•Necessaryforreproductionofnewcells
•MorethanoneDNApolymerasesexistindifferentorganisms
TaqDNApolymerase
•DerivedfromThermusaquaticus
•HeatstableDNApolymerase
•Idealtemperature72C
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8. Properties of DNA polymearse
•Needsapre-existingDNAtoduplicate
–CannotassembleanewstrandfromtemplateDNA
•CanonlyextendanexistingpieceofDNA
Calledprimers
3’ 5’
5’ 3’
•DNApolymeraseneedsMg
++
ascofactor
•EachDNApolymeraseworksbestunderoptimal
temperature,pHandsaltconcentration
PCRbufferprovidesoptimalpHandsaltcondition
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9. Properties of DNA polymearse
•DNA strands are anti-parallel
–One strand goes in 5’ 3’
–The complementary strand is opposite
•DNA polymerase always moves in one
direction (from 5’ 3’)
3’ 5’
5’ 3’
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10. Properties of DNA polymearse
•DNA polymerase incorporates the four
nucleotides (A, T, G, C) to the growing chain
•dNTP follow standard base pairing rule
3’ 5’
5’ 3’
dCTP
dTTP
dCTP
dGTPdATP
dGTP
dCTP
dTTP
dATP
dGTP
dCTP
dTTP
dATP
dATP
dGTPdCTPdTTPdATP dGTP
dATP
dGTP
dTTP
dATP
dCTP
dTTP
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11. The “Reaction” Components
1)TargetDNA-containsthesequencetobeamplified.
2)PairofPrimers-oligonucleotidesthatdefinethesequence
tobeamplified.
3)dNTPs-deoxynucleotidetriphosphates:DNAbuilding
blocks.
4)ThermostableDNAPolymerase-enzymethatcatalyzesthe
reaction
5)Mg
++
ions-cofactoroftheenzyme
6)Buffersolution–maintainspHandionicstrengthofthe
reactionsolutionsuitablefortheactivityoftheenzyme
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12. 1-DNA template
•DNA containing
region to be
sequenced
•Size of target DNA
to be amplified : up
to 3 Kb
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13. •A primer is a short synthetic oligonucleotide which is used in
many molecular techniques from PCRto DNA sequencing.
•Are designed to have a sequence which is the reverse
complement of a region of template or target DNA
2. Primer
18-24 bp best for general applications
•2setsofprimers
•Generally20-30nucleotideslong
•Syntheticallyproduced
•complimentarytothe3’endsof
targetDNA
•Notcomplimentarytoeachother
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14. 3-Enzyme
•Usually TaqPolymerase or anyone of the
natural or Recombinant thermostable
polymerases
•Stable at T
0
up to 95
0
C
•High procesivity
•TaqPol has 5’-3’ exoonly, no proofreading
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15. Typical PCR mix
In a thin wall Eppendorf tube assemble the following
PCR components Amount
Template DNA (5-200 ng)
1 mM dNTPs(200 uMfinal)
10 X PCR buffer
25 mM MgCl2 (1.5 mM final)
20 uMforward primer (20 pmolesfinal)
20 uMreverse primer (20 pmolesfinal)
5 units/uLTaq DNA polymerase (1.5 units)
Water
Final Volume
variable
10 uL
5 uL
3 uL
1 uL
1 uL
0.3 uL
Variable
50 uL
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16. Prepare a master mix for four rxns with a rxn volume of
25ul
component Stock sol Final sol Single PCR in
25ul
For 5 rxn
DNTP mix 1oomM 0.2mM
PCR Buffer10x 1x
FP 100mM 10mM
RP 100mM 10mM
MgCl2 25mM 1.5mM
TaqPoly 5u/ul 2.5
PCR Grade
H2O
- -
Tem. DNA 2ug/ul 2ug/ul
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17. The PCR Cycle
•A PCR machine controls temperature
•Typical PCR go through three steps
1-Denaturation:doublestrandedDNAsepratedintotwosingle
strands90-95c
2-Annealing:Coolingat40-60cPrimersattached
complementaryregionoftargetDNA
3-Extention:Heatingat70c.Theprimersextendedtoforma
newstrandofDNA
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18. Denaturation
•Heating separates the double
stranded DNA
–Denaturation
•Slow cooling anneals the two
strands
–Renaturation
Heat Cool
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19. Annealing
•Typicaltemperaturefrom50to60C
•PrimersattachedcomplementaryregionoftargetDNA
•Optimaltemperaturevariesbasedonprimerlength.
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20. Annealing Con’t…
•The annealing temp at which the primers
attach to template, can be calculated by
determining the melting temp (Tm) of primer
template hybrid. What is the TM of the ff
sequence?
5’ AGACTCAGAGAAACC 3’
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21. Extension
Optimal temperature 72C
DNA polymerase catalysesprimer extension as
complementary nucleotides are incorporated.
The newly generated DNA strands serve as template
DNA for the next cycle
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23. PCR Amplification
Exponential Amplification of template DNA
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24. PCR Amplification
•A 200ul of PCR mix has 100 template of DNA
molecule and the rxnwas performed for 10
cycle.
A) How many molecule of amplicons will be
generated
B) How many molecule amplicons will present in
0.1ul of rxn?
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25. Applications of PCR
•Classification
of organisms
•Genotyping
•Mutation
detection
•Sequencing
•Cancer research
•Detection of
pathogens
•DNA
fingerprinting
•Drug discovery
•Genetic
matching
•Genetic
engineering
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26. Applications Con’t
Primers can be created that will only bind and
amplify certain alleles of genes or mutations of
genes.
The role of PCR in diagnosis of infectious
diseases.
–Fastidious and slow growing microorganism
–Detect antimicrobial resistance
–Detect microorganism cannot be cultivated
–Measurement value of viral load
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27. Detection Of Pathogens
Sensitivityof detection of PCR-
amplified M. tuberculosisDNA.
(Kaul et al.1994)
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Detection of ACEgene on 2% agarose gel
electrophoresis.Lane 1. Show 100bp DNA
marker. Lane 2 and 5 show homozygous DD.
Lane 3 and 4-show heterozygous II genotype.
Lane 6 shows control.

28. PCR Vs DNAReplication
PCRisaninvitromethodofDNAamplificationinwhich
thousandstomillionsofcopiesofDNAareproduced.
DNAReplicationisanaturalprocessthatproducestwo
identicalcopiesofDNAfromoneDNAmolecule.
Steps
PCRhasthreesteps;denaturation,primerannealingandstrand
extension.
DNAReplicationhasthreesteps;initiation,elongationand
termination.
Involvement of Primers
PCRneedsartificialprimers DNAReplicationdoesnotneedartificialprimers.Ashort
fragmentofRNAisinvolvedinDNAreplication.
Denaturing of the double-Strands
Doublestrandsareseparatedbyapplyingahightemperaturein
PCR.
Doublestrandsareseparatedfromeachotherbythe
enzymeDNAhelicaseinDNAReplication.
Enzyme Involved
PCRusesTaqpolymerase. DNAReplicationusesDNApolymerase.
Temperature
PCRoccursatthreedifferenttemperaturesinsideamachine. DNAReplicationoccursatbodytemperaturewithinthe
bodyofthelivingorganism.
In vivo or In vitro
PCRisaninvitromethod. DNAReplicationisaninvivomethod.
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29. Summary
blood, chorionic
villus, amniotic
fluid, semen, hair
root, saliva
68,719,476,736 copies
Gel Analysis,
Restriction
Digestion,
Sequencing
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30. Thank you