the polymerase chain reaction (pcr) by dr. naglaa fathy lecturer of biochemistry & molecular...
TRANSCRIPT
The Polymerase ChainThe Polymerase ChainReaction (PCR)Reaction (PCR)
ByBy
Dr NAGLAA FATHY Dr NAGLAA FATHY
Lecturer of Biochemistry amp Molecular Biology Lecturer of Biochemistry amp Molecular Biology Faculty of Medicine Faculty of Medicine Benha University Benha University
E-mail E-mail naglaa_fathy722000yahoocomnaglaa_fathy722000yahoocom NaglaalhusseinifmedbueduegNaglaalhusseinifmedbuedueg
What is the PolymeraseWhat is the PolymeraseChain ReactionChain Reaction
Itrsquos a means of selectively amplifying aItrsquos a means of selectively amplifying a
particular segment of DNAparticular segment of DNA The segment may represent a small part The segment may represent a small part
of a large and complex mixture of DNAsof a large and complex mixture of DNAs
Invented by Kary MullisInvented by Kary Mullis
Mullis and Faloona 1987 SpecificMullis and Faloona 1987 Specific
synthesis of DNA in vitro via asynthesis of DNA in vitro via a
polymerase-catalyzed chain reactionpolymerase-catalyzed chain reaction
Nobel Prize 1993Nobel Prize 1993
Kary MullisKary Mullis
Did He Really Invent PCRDid He Really Invent PCR
bull bull The basic principle of The basic principle of replicating a piece of DNA replicating a piece of DNA using two primers had already using two primers had already been described by Gobind been described by Gobind Khorana in 1971ndash Kleppe Khorana in 1971ndash Kleppe et et al al (1971) J Mol Biol 56 (1971) J Mol Biol 56 341-346341-346
bull bull Progress was limited by primer Progress was limited by primer synthesis and polymerase synthesis and polymerase purification issuespurification issues
bull bull Mullis properly exploited Mullis properly exploited amplificationamplification
PCRPCR
Specifically targets and amplifies aSpecifically targets and amplifies a
SINGLE sequence from within a complexSINGLE sequence from within a complex
mixture of DNAmixture of DNA
How is this different from cloningHow is this different from cloning
Amplify DNAAmplify DNAPCRPCR
In vitro amplification (in a test tube)In vitro amplification (in a test tube) Enzymatic Taq polymeraseEnzymatic Taq polymerase
ndash ndash Temperature-resistant DNATemperature-resistant DNA
polymerase ( Thermus aquaticus)polymerase ( Thermus aquaticus) Heat resistantHeat resistant Best for lt2 kb targetBest for lt2 kb target
Takes advantage of basicTakes advantage of basicrequirements of replicationrequirements of replication
A DNA templateA DNA template NucleotidesNucleotides PrimersPrimers polymerasepolymerase
PCR is DNA replication in a test tubePCR is DNA replication in a test tube
PRIMERSPRIMERS
Primers short ssDNA sequencescomplementary to border of sequence of interest
PrimersPrimers
Must have some information Must have some information about sequence flanking your about sequence flanking your targettargetPrimers provide specificityPrimers provide specificity
ends pointing towards each otherends pointing towards each other Complementary to opposite strands with Complementary to opposite strands with
3rsquo3rsquo Should have similar melting temperaturesShould have similar melting temperatures
PrimersPrimers
PCRPCRRegion of interestbetween primers
2 Anneal
3 Extend
Taq polymerase enzymatic extension
PCRRepeated Cycles ofRepeated Cycles of
1 Denaturation 1 Denaturation
2 Annealing2 Annealing
3 Extention 3 Extention
1Denaturation
2 Anneal
3 Extend
Melting temperatureMelting temperature
TTmmooC Temperature at whichC Temperature at which half possible H bonds arehalf possible H bonds are formedformedTTmmooC = 2(A+T) + 4(G+C)C = 2(A+T) + 4(G+C)
55 - -AGACTCAGAGAGAACCC-3AGACTCAGAGAGAACCC-3
4Gs 5Cs 4Gs 5Cs 7As 1T7As 1T
TTmmooC=C= (4x9) + (2x8) = 36+16 = (4x9) + (2x8) = 36+16 = 525200CC
Annealing T =TAnnealing T =Tmm00C -5C -5
Heat-stable polymerase is vitalHeat-stable polymerase is vitalto the ease of the processto the ease of the processhelliphellip
Thermus aquaticusThermus aquaticus
Thermus aquaticus from hot springs inYellowstone National Park USA
The ThermusThe Thermusaquaticus DNAaquaticus DNA
polymerasepolymerase
TaqTaq Not permanentlyNot permanently
destroyed at 94ordmCdestroyed at 94ordmC OptimalOptimal
temperature is 72ordmCtemperature is 72ordmC
Problems with TaqProblems with Taq
Taq DNA polymerase - thermostableTaq DNA polymerase - thermostable Lack of 3prime-5prime exonuclease ndash proofreadingLack of 3prime-5prime exonuclease ndash proofreading
Error rate = 2 times 10Error rate = 2 times 10-4-4 nucleotdescycle nucleotdescycle Newer polymerases have high fidelityNewer polymerases have high fidelity
High fidelity polymerase - HiFi TaqHigh fidelity polymerase - HiFi Taq
Termplates for PCRTermplates for PCR
Small amount of templateSmall amount of template In theory a single moleculeIn theory a single molecule Do not need to isolate sequence of interestDo not need to isolate sequence of interest DNA template need not be highly purifiedDNA template need not be highly purified DNA is stable in absence of nucleasesDNA is stable in absence of nucleases
Templates for PCRTemplates for PCR
1048708 1048708 Dried bloodDried blood1048708 1048708 Semen stainsSemen stains1048708 1048708 Vaginal swabsVaginal swabs1048708 1048708 Single hairSingle hair1048708 1048708 Finger nail scrapingsFinger nail scrapings1048708 1048708 Egyptian mummiesEgyptian mummies1048708 1048708 Buccal SwabBuccal Swab1048708 1048708 Tooth brushesTooth brushes
Basic reactionBasic reaction
Thermocycling PCR machine1048707 Previously ndash need to overlay oil to prevent evaporation1048707 Automatically Change temperature1048707 Temperature gradient
The Basics of PCR CyclingThe Basics of PCR Cycling
bull bull 30ndash35 cycles each30ndash35 cycles each
comprisingcomprising
ndash ndash denaturation (95degC)denaturation (95degC)
30 sec30 sec
ndash ndash annealing (55ndash60degC)annealing (55ndash60degC)
30 sec30 sec
ndash ndash extension (72degC)extension (72degC)
time depends ontime depends on
product sizeproduct size
How many copiesHow many copies
bull bull No target products are made until the third No target products are made until the third cyclecycle
bull bull The accumulation is not strictly a doublingThe accumulation is not strictly a doubling
at each cycle in the early phaseat each cycle in the early phase
bull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 target
copies (~1times10copies (~1times1099))
How many cyclesHow many cycles
bull bull Increasing the cycleIncreasing the cyclenumber above ~35 hasnumber above ~35 haslittle positive effectlittle positive effectbull bull The plateau occurs whenThe plateau occurs whenndash ndash The reagents are depletedThe reagents are depletedndash ndash The products re-annealThe products re-annealndash ndash The polymerase isThe polymerase isdamageddamaged- Unwanted products- Unwanted productsaccumulateaccumulate
Basic reactionBasic reaction Oligonucleotide Oligonucleotide
primersprimers Design to flank the Design to flank the
desired sequencedesired sequence Steps include (30-Steps include (30-
40 steps)40 steps) 1048708 1048708 Denaturation at Denaturation at
94degC94degC 1048708 1048708 Primer annealing Primer annealing
at Tm-5degCat Tm-5degC 1048708 1048708 Extension at 72degCExtension at 72degC
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
What is the PolymeraseWhat is the PolymeraseChain ReactionChain Reaction
Itrsquos a means of selectively amplifying aItrsquos a means of selectively amplifying a
particular segment of DNAparticular segment of DNA The segment may represent a small part The segment may represent a small part
of a large and complex mixture of DNAsof a large and complex mixture of DNAs
Invented by Kary MullisInvented by Kary Mullis
Mullis and Faloona 1987 SpecificMullis and Faloona 1987 Specific
synthesis of DNA in vitro via asynthesis of DNA in vitro via a
polymerase-catalyzed chain reactionpolymerase-catalyzed chain reaction
Nobel Prize 1993Nobel Prize 1993
Kary MullisKary Mullis
Did He Really Invent PCRDid He Really Invent PCR
bull bull The basic principle of The basic principle of replicating a piece of DNA replicating a piece of DNA using two primers had already using two primers had already been described by Gobind been described by Gobind Khorana in 1971ndash Kleppe Khorana in 1971ndash Kleppe et et al al (1971) J Mol Biol 56 (1971) J Mol Biol 56 341-346341-346
bull bull Progress was limited by primer Progress was limited by primer synthesis and polymerase synthesis and polymerase purification issuespurification issues
bull bull Mullis properly exploited Mullis properly exploited amplificationamplification
PCRPCR
Specifically targets and amplifies aSpecifically targets and amplifies a
SINGLE sequence from within a complexSINGLE sequence from within a complex
mixture of DNAmixture of DNA
How is this different from cloningHow is this different from cloning
Amplify DNAAmplify DNAPCRPCR
In vitro amplification (in a test tube)In vitro amplification (in a test tube) Enzymatic Taq polymeraseEnzymatic Taq polymerase
ndash ndash Temperature-resistant DNATemperature-resistant DNA
polymerase ( Thermus aquaticus)polymerase ( Thermus aquaticus) Heat resistantHeat resistant Best for lt2 kb targetBest for lt2 kb target
Takes advantage of basicTakes advantage of basicrequirements of replicationrequirements of replication
A DNA templateA DNA template NucleotidesNucleotides PrimersPrimers polymerasepolymerase
PCR is DNA replication in a test tubePCR is DNA replication in a test tube
PRIMERSPRIMERS
Primers short ssDNA sequencescomplementary to border of sequence of interest
PrimersPrimers
Must have some information Must have some information about sequence flanking your about sequence flanking your targettargetPrimers provide specificityPrimers provide specificity
ends pointing towards each otherends pointing towards each other Complementary to opposite strands with Complementary to opposite strands with
3rsquo3rsquo Should have similar melting temperaturesShould have similar melting temperatures
PrimersPrimers
PCRPCRRegion of interestbetween primers
2 Anneal
3 Extend
Taq polymerase enzymatic extension
PCRRepeated Cycles ofRepeated Cycles of
1 Denaturation 1 Denaturation
2 Annealing2 Annealing
3 Extention 3 Extention
1Denaturation
2 Anneal
3 Extend
Melting temperatureMelting temperature
TTmmooC Temperature at whichC Temperature at which half possible H bonds arehalf possible H bonds are formedformedTTmmooC = 2(A+T) + 4(G+C)C = 2(A+T) + 4(G+C)
55 - -AGACTCAGAGAGAACCC-3AGACTCAGAGAGAACCC-3
4Gs 5Cs 4Gs 5Cs 7As 1T7As 1T
TTmmooC=C= (4x9) + (2x8) = 36+16 = (4x9) + (2x8) = 36+16 = 525200CC
Annealing T =TAnnealing T =Tmm00C -5C -5
Heat-stable polymerase is vitalHeat-stable polymerase is vitalto the ease of the processto the ease of the processhelliphellip
Thermus aquaticusThermus aquaticus
Thermus aquaticus from hot springs inYellowstone National Park USA
The ThermusThe Thermusaquaticus DNAaquaticus DNA
polymerasepolymerase
TaqTaq Not permanentlyNot permanently
destroyed at 94ordmCdestroyed at 94ordmC OptimalOptimal
temperature is 72ordmCtemperature is 72ordmC
Problems with TaqProblems with Taq
Taq DNA polymerase - thermostableTaq DNA polymerase - thermostable Lack of 3prime-5prime exonuclease ndash proofreadingLack of 3prime-5prime exonuclease ndash proofreading
Error rate = 2 times 10Error rate = 2 times 10-4-4 nucleotdescycle nucleotdescycle Newer polymerases have high fidelityNewer polymerases have high fidelity
High fidelity polymerase - HiFi TaqHigh fidelity polymerase - HiFi Taq
Termplates for PCRTermplates for PCR
Small amount of templateSmall amount of template In theory a single moleculeIn theory a single molecule Do not need to isolate sequence of interestDo not need to isolate sequence of interest DNA template need not be highly purifiedDNA template need not be highly purified DNA is stable in absence of nucleasesDNA is stable in absence of nucleases
Templates for PCRTemplates for PCR
1048708 1048708 Dried bloodDried blood1048708 1048708 Semen stainsSemen stains1048708 1048708 Vaginal swabsVaginal swabs1048708 1048708 Single hairSingle hair1048708 1048708 Finger nail scrapingsFinger nail scrapings1048708 1048708 Egyptian mummiesEgyptian mummies1048708 1048708 Buccal SwabBuccal Swab1048708 1048708 Tooth brushesTooth brushes
Basic reactionBasic reaction
Thermocycling PCR machine1048707 Previously ndash need to overlay oil to prevent evaporation1048707 Automatically Change temperature1048707 Temperature gradient
The Basics of PCR CyclingThe Basics of PCR Cycling
bull bull 30ndash35 cycles each30ndash35 cycles each
comprisingcomprising
ndash ndash denaturation (95degC)denaturation (95degC)
30 sec30 sec
ndash ndash annealing (55ndash60degC)annealing (55ndash60degC)
30 sec30 sec
ndash ndash extension (72degC)extension (72degC)
time depends ontime depends on
product sizeproduct size
How many copiesHow many copies
bull bull No target products are made until the third No target products are made until the third cyclecycle
bull bull The accumulation is not strictly a doublingThe accumulation is not strictly a doubling
at each cycle in the early phaseat each cycle in the early phase
bull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 target
copies (~1times10copies (~1times1099))
How many cyclesHow many cycles
bull bull Increasing the cycleIncreasing the cyclenumber above ~35 hasnumber above ~35 haslittle positive effectlittle positive effectbull bull The plateau occurs whenThe plateau occurs whenndash ndash The reagents are depletedThe reagents are depletedndash ndash The products re-annealThe products re-annealndash ndash The polymerase isThe polymerase isdamageddamaged- Unwanted products- Unwanted productsaccumulateaccumulate
Basic reactionBasic reaction Oligonucleotide Oligonucleotide
primersprimers Design to flank the Design to flank the
desired sequencedesired sequence Steps include (30-Steps include (30-
40 steps)40 steps) 1048708 1048708 Denaturation at Denaturation at
94degC94degC 1048708 1048708 Primer annealing Primer annealing
at Tm-5degCat Tm-5degC 1048708 1048708 Extension at 72degCExtension at 72degC
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
Invented by Kary MullisInvented by Kary Mullis
Mullis and Faloona 1987 SpecificMullis and Faloona 1987 Specific
synthesis of DNA in vitro via asynthesis of DNA in vitro via a
polymerase-catalyzed chain reactionpolymerase-catalyzed chain reaction
Nobel Prize 1993Nobel Prize 1993
Kary MullisKary Mullis
Did He Really Invent PCRDid He Really Invent PCR
bull bull The basic principle of The basic principle of replicating a piece of DNA replicating a piece of DNA using two primers had already using two primers had already been described by Gobind been described by Gobind Khorana in 1971ndash Kleppe Khorana in 1971ndash Kleppe et et al al (1971) J Mol Biol 56 (1971) J Mol Biol 56 341-346341-346
bull bull Progress was limited by primer Progress was limited by primer synthesis and polymerase synthesis and polymerase purification issuespurification issues
bull bull Mullis properly exploited Mullis properly exploited amplificationamplification
PCRPCR
Specifically targets and amplifies aSpecifically targets and amplifies a
SINGLE sequence from within a complexSINGLE sequence from within a complex
mixture of DNAmixture of DNA
How is this different from cloningHow is this different from cloning
Amplify DNAAmplify DNAPCRPCR
In vitro amplification (in a test tube)In vitro amplification (in a test tube) Enzymatic Taq polymeraseEnzymatic Taq polymerase
ndash ndash Temperature-resistant DNATemperature-resistant DNA
polymerase ( Thermus aquaticus)polymerase ( Thermus aquaticus) Heat resistantHeat resistant Best for lt2 kb targetBest for lt2 kb target
Takes advantage of basicTakes advantage of basicrequirements of replicationrequirements of replication
A DNA templateA DNA template NucleotidesNucleotides PrimersPrimers polymerasepolymerase
PCR is DNA replication in a test tubePCR is DNA replication in a test tube
PRIMERSPRIMERS
Primers short ssDNA sequencescomplementary to border of sequence of interest
PrimersPrimers
Must have some information Must have some information about sequence flanking your about sequence flanking your targettargetPrimers provide specificityPrimers provide specificity
ends pointing towards each otherends pointing towards each other Complementary to opposite strands with Complementary to opposite strands with
3rsquo3rsquo Should have similar melting temperaturesShould have similar melting temperatures
PrimersPrimers
PCRPCRRegion of interestbetween primers
2 Anneal
3 Extend
Taq polymerase enzymatic extension
PCRRepeated Cycles ofRepeated Cycles of
1 Denaturation 1 Denaturation
2 Annealing2 Annealing
3 Extention 3 Extention
1Denaturation
2 Anneal
3 Extend
Melting temperatureMelting temperature
TTmmooC Temperature at whichC Temperature at which half possible H bonds arehalf possible H bonds are formedformedTTmmooC = 2(A+T) + 4(G+C)C = 2(A+T) + 4(G+C)
55 - -AGACTCAGAGAGAACCC-3AGACTCAGAGAGAACCC-3
4Gs 5Cs 4Gs 5Cs 7As 1T7As 1T
TTmmooC=C= (4x9) + (2x8) = 36+16 = (4x9) + (2x8) = 36+16 = 525200CC
Annealing T =TAnnealing T =Tmm00C -5C -5
Heat-stable polymerase is vitalHeat-stable polymerase is vitalto the ease of the processto the ease of the processhelliphellip
Thermus aquaticusThermus aquaticus
Thermus aquaticus from hot springs inYellowstone National Park USA
The ThermusThe Thermusaquaticus DNAaquaticus DNA
polymerasepolymerase
TaqTaq Not permanentlyNot permanently
destroyed at 94ordmCdestroyed at 94ordmC OptimalOptimal
temperature is 72ordmCtemperature is 72ordmC
Problems with TaqProblems with Taq
Taq DNA polymerase - thermostableTaq DNA polymerase - thermostable Lack of 3prime-5prime exonuclease ndash proofreadingLack of 3prime-5prime exonuclease ndash proofreading
Error rate = 2 times 10Error rate = 2 times 10-4-4 nucleotdescycle nucleotdescycle Newer polymerases have high fidelityNewer polymerases have high fidelity
High fidelity polymerase - HiFi TaqHigh fidelity polymerase - HiFi Taq
Termplates for PCRTermplates for PCR
Small amount of templateSmall amount of template In theory a single moleculeIn theory a single molecule Do not need to isolate sequence of interestDo not need to isolate sequence of interest DNA template need not be highly purifiedDNA template need not be highly purified DNA is stable in absence of nucleasesDNA is stable in absence of nucleases
Templates for PCRTemplates for PCR
1048708 1048708 Dried bloodDried blood1048708 1048708 Semen stainsSemen stains1048708 1048708 Vaginal swabsVaginal swabs1048708 1048708 Single hairSingle hair1048708 1048708 Finger nail scrapingsFinger nail scrapings1048708 1048708 Egyptian mummiesEgyptian mummies1048708 1048708 Buccal SwabBuccal Swab1048708 1048708 Tooth brushesTooth brushes
Basic reactionBasic reaction
Thermocycling PCR machine1048707 Previously ndash need to overlay oil to prevent evaporation1048707 Automatically Change temperature1048707 Temperature gradient
The Basics of PCR CyclingThe Basics of PCR Cycling
bull bull 30ndash35 cycles each30ndash35 cycles each
comprisingcomprising
ndash ndash denaturation (95degC)denaturation (95degC)
30 sec30 sec
ndash ndash annealing (55ndash60degC)annealing (55ndash60degC)
30 sec30 sec
ndash ndash extension (72degC)extension (72degC)
time depends ontime depends on
product sizeproduct size
How many copiesHow many copies
bull bull No target products are made until the third No target products are made until the third cyclecycle
bull bull The accumulation is not strictly a doublingThe accumulation is not strictly a doubling
at each cycle in the early phaseat each cycle in the early phase
bull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 target
copies (~1times10copies (~1times1099))
How many cyclesHow many cycles
bull bull Increasing the cycleIncreasing the cyclenumber above ~35 hasnumber above ~35 haslittle positive effectlittle positive effectbull bull The plateau occurs whenThe plateau occurs whenndash ndash The reagents are depletedThe reagents are depletedndash ndash The products re-annealThe products re-annealndash ndash The polymerase isThe polymerase isdamageddamaged- Unwanted products- Unwanted productsaccumulateaccumulate
Basic reactionBasic reaction Oligonucleotide Oligonucleotide
primersprimers Design to flank the Design to flank the
desired sequencedesired sequence Steps include (30-Steps include (30-
40 steps)40 steps) 1048708 1048708 Denaturation at Denaturation at
94degC94degC 1048708 1048708 Primer annealing Primer annealing
at Tm-5degCat Tm-5degC 1048708 1048708 Extension at 72degCExtension at 72degC
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
Kary MullisKary Mullis
Did He Really Invent PCRDid He Really Invent PCR
bull bull The basic principle of The basic principle of replicating a piece of DNA replicating a piece of DNA using two primers had already using two primers had already been described by Gobind been described by Gobind Khorana in 1971ndash Kleppe Khorana in 1971ndash Kleppe et et al al (1971) J Mol Biol 56 (1971) J Mol Biol 56 341-346341-346
bull bull Progress was limited by primer Progress was limited by primer synthesis and polymerase synthesis and polymerase purification issuespurification issues
bull bull Mullis properly exploited Mullis properly exploited amplificationamplification
PCRPCR
Specifically targets and amplifies aSpecifically targets and amplifies a
SINGLE sequence from within a complexSINGLE sequence from within a complex
mixture of DNAmixture of DNA
How is this different from cloningHow is this different from cloning
Amplify DNAAmplify DNAPCRPCR
In vitro amplification (in a test tube)In vitro amplification (in a test tube) Enzymatic Taq polymeraseEnzymatic Taq polymerase
ndash ndash Temperature-resistant DNATemperature-resistant DNA
polymerase ( Thermus aquaticus)polymerase ( Thermus aquaticus) Heat resistantHeat resistant Best for lt2 kb targetBest for lt2 kb target
Takes advantage of basicTakes advantage of basicrequirements of replicationrequirements of replication
A DNA templateA DNA template NucleotidesNucleotides PrimersPrimers polymerasepolymerase
PCR is DNA replication in a test tubePCR is DNA replication in a test tube
PRIMERSPRIMERS
Primers short ssDNA sequencescomplementary to border of sequence of interest
PrimersPrimers
Must have some information Must have some information about sequence flanking your about sequence flanking your targettargetPrimers provide specificityPrimers provide specificity
ends pointing towards each otherends pointing towards each other Complementary to opposite strands with Complementary to opposite strands with
3rsquo3rsquo Should have similar melting temperaturesShould have similar melting temperatures
PrimersPrimers
PCRPCRRegion of interestbetween primers
2 Anneal
3 Extend
Taq polymerase enzymatic extension
PCRRepeated Cycles ofRepeated Cycles of
1 Denaturation 1 Denaturation
2 Annealing2 Annealing
3 Extention 3 Extention
1Denaturation
2 Anneal
3 Extend
Melting temperatureMelting temperature
TTmmooC Temperature at whichC Temperature at which half possible H bonds arehalf possible H bonds are formedformedTTmmooC = 2(A+T) + 4(G+C)C = 2(A+T) + 4(G+C)
55 - -AGACTCAGAGAGAACCC-3AGACTCAGAGAGAACCC-3
4Gs 5Cs 4Gs 5Cs 7As 1T7As 1T
TTmmooC=C= (4x9) + (2x8) = 36+16 = (4x9) + (2x8) = 36+16 = 525200CC
Annealing T =TAnnealing T =Tmm00C -5C -5
Heat-stable polymerase is vitalHeat-stable polymerase is vitalto the ease of the processto the ease of the processhelliphellip
Thermus aquaticusThermus aquaticus
Thermus aquaticus from hot springs inYellowstone National Park USA
The ThermusThe Thermusaquaticus DNAaquaticus DNA
polymerasepolymerase
TaqTaq Not permanentlyNot permanently
destroyed at 94ordmCdestroyed at 94ordmC OptimalOptimal
temperature is 72ordmCtemperature is 72ordmC
Problems with TaqProblems with Taq
Taq DNA polymerase - thermostableTaq DNA polymerase - thermostable Lack of 3prime-5prime exonuclease ndash proofreadingLack of 3prime-5prime exonuclease ndash proofreading
Error rate = 2 times 10Error rate = 2 times 10-4-4 nucleotdescycle nucleotdescycle Newer polymerases have high fidelityNewer polymerases have high fidelity
High fidelity polymerase - HiFi TaqHigh fidelity polymerase - HiFi Taq
Termplates for PCRTermplates for PCR
Small amount of templateSmall amount of template In theory a single moleculeIn theory a single molecule Do not need to isolate sequence of interestDo not need to isolate sequence of interest DNA template need not be highly purifiedDNA template need not be highly purified DNA is stable in absence of nucleasesDNA is stable in absence of nucleases
Templates for PCRTemplates for PCR
1048708 1048708 Dried bloodDried blood1048708 1048708 Semen stainsSemen stains1048708 1048708 Vaginal swabsVaginal swabs1048708 1048708 Single hairSingle hair1048708 1048708 Finger nail scrapingsFinger nail scrapings1048708 1048708 Egyptian mummiesEgyptian mummies1048708 1048708 Buccal SwabBuccal Swab1048708 1048708 Tooth brushesTooth brushes
Basic reactionBasic reaction
Thermocycling PCR machine1048707 Previously ndash need to overlay oil to prevent evaporation1048707 Automatically Change temperature1048707 Temperature gradient
The Basics of PCR CyclingThe Basics of PCR Cycling
bull bull 30ndash35 cycles each30ndash35 cycles each
comprisingcomprising
ndash ndash denaturation (95degC)denaturation (95degC)
30 sec30 sec
ndash ndash annealing (55ndash60degC)annealing (55ndash60degC)
30 sec30 sec
ndash ndash extension (72degC)extension (72degC)
time depends ontime depends on
product sizeproduct size
How many copiesHow many copies
bull bull No target products are made until the third No target products are made until the third cyclecycle
bull bull The accumulation is not strictly a doublingThe accumulation is not strictly a doubling
at each cycle in the early phaseat each cycle in the early phase
bull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 target
copies (~1times10copies (~1times1099))
How many cyclesHow many cycles
bull bull Increasing the cycleIncreasing the cyclenumber above ~35 hasnumber above ~35 haslittle positive effectlittle positive effectbull bull The plateau occurs whenThe plateau occurs whenndash ndash The reagents are depletedThe reagents are depletedndash ndash The products re-annealThe products re-annealndash ndash The polymerase isThe polymerase isdamageddamaged- Unwanted products- Unwanted productsaccumulateaccumulate
Basic reactionBasic reaction Oligonucleotide Oligonucleotide
primersprimers Design to flank the Design to flank the
desired sequencedesired sequence Steps include (30-Steps include (30-
40 steps)40 steps) 1048708 1048708 Denaturation at Denaturation at
94degC94degC 1048708 1048708 Primer annealing Primer annealing
at Tm-5degCat Tm-5degC 1048708 1048708 Extension at 72degCExtension at 72degC
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
Did He Really Invent PCRDid He Really Invent PCR
bull bull The basic principle of The basic principle of replicating a piece of DNA replicating a piece of DNA using two primers had already using two primers had already been described by Gobind been described by Gobind Khorana in 1971ndash Kleppe Khorana in 1971ndash Kleppe et et al al (1971) J Mol Biol 56 (1971) J Mol Biol 56 341-346341-346
bull bull Progress was limited by primer Progress was limited by primer synthesis and polymerase synthesis and polymerase purification issuespurification issues
bull bull Mullis properly exploited Mullis properly exploited amplificationamplification
PCRPCR
Specifically targets and amplifies aSpecifically targets and amplifies a
SINGLE sequence from within a complexSINGLE sequence from within a complex
mixture of DNAmixture of DNA
How is this different from cloningHow is this different from cloning
Amplify DNAAmplify DNAPCRPCR
In vitro amplification (in a test tube)In vitro amplification (in a test tube) Enzymatic Taq polymeraseEnzymatic Taq polymerase
ndash ndash Temperature-resistant DNATemperature-resistant DNA
polymerase ( Thermus aquaticus)polymerase ( Thermus aquaticus) Heat resistantHeat resistant Best for lt2 kb targetBest for lt2 kb target
Takes advantage of basicTakes advantage of basicrequirements of replicationrequirements of replication
A DNA templateA DNA template NucleotidesNucleotides PrimersPrimers polymerasepolymerase
PCR is DNA replication in a test tubePCR is DNA replication in a test tube
PRIMERSPRIMERS
Primers short ssDNA sequencescomplementary to border of sequence of interest
PrimersPrimers
Must have some information Must have some information about sequence flanking your about sequence flanking your targettargetPrimers provide specificityPrimers provide specificity
ends pointing towards each otherends pointing towards each other Complementary to opposite strands with Complementary to opposite strands with
3rsquo3rsquo Should have similar melting temperaturesShould have similar melting temperatures
PrimersPrimers
PCRPCRRegion of interestbetween primers
2 Anneal
3 Extend
Taq polymerase enzymatic extension
PCRRepeated Cycles ofRepeated Cycles of
1 Denaturation 1 Denaturation
2 Annealing2 Annealing
3 Extention 3 Extention
1Denaturation
2 Anneal
3 Extend
Melting temperatureMelting temperature
TTmmooC Temperature at whichC Temperature at which half possible H bonds arehalf possible H bonds are formedformedTTmmooC = 2(A+T) + 4(G+C)C = 2(A+T) + 4(G+C)
55 - -AGACTCAGAGAGAACCC-3AGACTCAGAGAGAACCC-3
4Gs 5Cs 4Gs 5Cs 7As 1T7As 1T
TTmmooC=C= (4x9) + (2x8) = 36+16 = (4x9) + (2x8) = 36+16 = 525200CC
Annealing T =TAnnealing T =Tmm00C -5C -5
Heat-stable polymerase is vitalHeat-stable polymerase is vitalto the ease of the processto the ease of the processhelliphellip
Thermus aquaticusThermus aquaticus
Thermus aquaticus from hot springs inYellowstone National Park USA
The ThermusThe Thermusaquaticus DNAaquaticus DNA
polymerasepolymerase
TaqTaq Not permanentlyNot permanently
destroyed at 94ordmCdestroyed at 94ordmC OptimalOptimal
temperature is 72ordmCtemperature is 72ordmC
Problems with TaqProblems with Taq
Taq DNA polymerase - thermostableTaq DNA polymerase - thermostable Lack of 3prime-5prime exonuclease ndash proofreadingLack of 3prime-5prime exonuclease ndash proofreading
Error rate = 2 times 10Error rate = 2 times 10-4-4 nucleotdescycle nucleotdescycle Newer polymerases have high fidelityNewer polymerases have high fidelity
High fidelity polymerase - HiFi TaqHigh fidelity polymerase - HiFi Taq
Termplates for PCRTermplates for PCR
Small amount of templateSmall amount of template In theory a single moleculeIn theory a single molecule Do not need to isolate sequence of interestDo not need to isolate sequence of interest DNA template need not be highly purifiedDNA template need not be highly purified DNA is stable in absence of nucleasesDNA is stable in absence of nucleases
Templates for PCRTemplates for PCR
1048708 1048708 Dried bloodDried blood1048708 1048708 Semen stainsSemen stains1048708 1048708 Vaginal swabsVaginal swabs1048708 1048708 Single hairSingle hair1048708 1048708 Finger nail scrapingsFinger nail scrapings1048708 1048708 Egyptian mummiesEgyptian mummies1048708 1048708 Buccal SwabBuccal Swab1048708 1048708 Tooth brushesTooth brushes
Basic reactionBasic reaction
Thermocycling PCR machine1048707 Previously ndash need to overlay oil to prevent evaporation1048707 Automatically Change temperature1048707 Temperature gradient
The Basics of PCR CyclingThe Basics of PCR Cycling
bull bull 30ndash35 cycles each30ndash35 cycles each
comprisingcomprising
ndash ndash denaturation (95degC)denaturation (95degC)
30 sec30 sec
ndash ndash annealing (55ndash60degC)annealing (55ndash60degC)
30 sec30 sec
ndash ndash extension (72degC)extension (72degC)
time depends ontime depends on
product sizeproduct size
How many copiesHow many copies
bull bull No target products are made until the third No target products are made until the third cyclecycle
bull bull The accumulation is not strictly a doublingThe accumulation is not strictly a doubling
at each cycle in the early phaseat each cycle in the early phase
bull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 target
copies (~1times10copies (~1times1099))
How many cyclesHow many cycles
bull bull Increasing the cycleIncreasing the cyclenumber above ~35 hasnumber above ~35 haslittle positive effectlittle positive effectbull bull The plateau occurs whenThe plateau occurs whenndash ndash The reagents are depletedThe reagents are depletedndash ndash The products re-annealThe products re-annealndash ndash The polymerase isThe polymerase isdamageddamaged- Unwanted products- Unwanted productsaccumulateaccumulate
Basic reactionBasic reaction Oligonucleotide Oligonucleotide
primersprimers Design to flank the Design to flank the
desired sequencedesired sequence Steps include (30-Steps include (30-
40 steps)40 steps) 1048708 1048708 Denaturation at Denaturation at
94degC94degC 1048708 1048708 Primer annealing Primer annealing
at Tm-5degCat Tm-5degC 1048708 1048708 Extension at 72degCExtension at 72degC
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
PCRPCR
Specifically targets and amplifies aSpecifically targets and amplifies a
SINGLE sequence from within a complexSINGLE sequence from within a complex
mixture of DNAmixture of DNA
How is this different from cloningHow is this different from cloning
Amplify DNAAmplify DNAPCRPCR
In vitro amplification (in a test tube)In vitro amplification (in a test tube) Enzymatic Taq polymeraseEnzymatic Taq polymerase
ndash ndash Temperature-resistant DNATemperature-resistant DNA
polymerase ( Thermus aquaticus)polymerase ( Thermus aquaticus) Heat resistantHeat resistant Best for lt2 kb targetBest for lt2 kb target
Takes advantage of basicTakes advantage of basicrequirements of replicationrequirements of replication
A DNA templateA DNA template NucleotidesNucleotides PrimersPrimers polymerasepolymerase
PCR is DNA replication in a test tubePCR is DNA replication in a test tube
PRIMERSPRIMERS
Primers short ssDNA sequencescomplementary to border of sequence of interest
PrimersPrimers
Must have some information Must have some information about sequence flanking your about sequence flanking your targettargetPrimers provide specificityPrimers provide specificity
ends pointing towards each otherends pointing towards each other Complementary to opposite strands with Complementary to opposite strands with
3rsquo3rsquo Should have similar melting temperaturesShould have similar melting temperatures
PrimersPrimers
PCRPCRRegion of interestbetween primers
2 Anneal
3 Extend
Taq polymerase enzymatic extension
PCRRepeated Cycles ofRepeated Cycles of
1 Denaturation 1 Denaturation
2 Annealing2 Annealing
3 Extention 3 Extention
1Denaturation
2 Anneal
3 Extend
Melting temperatureMelting temperature
TTmmooC Temperature at whichC Temperature at which half possible H bonds arehalf possible H bonds are formedformedTTmmooC = 2(A+T) + 4(G+C)C = 2(A+T) + 4(G+C)
55 - -AGACTCAGAGAGAACCC-3AGACTCAGAGAGAACCC-3
4Gs 5Cs 4Gs 5Cs 7As 1T7As 1T
TTmmooC=C= (4x9) + (2x8) = 36+16 = (4x9) + (2x8) = 36+16 = 525200CC
Annealing T =TAnnealing T =Tmm00C -5C -5
Heat-stable polymerase is vitalHeat-stable polymerase is vitalto the ease of the processto the ease of the processhelliphellip
Thermus aquaticusThermus aquaticus
Thermus aquaticus from hot springs inYellowstone National Park USA
The ThermusThe Thermusaquaticus DNAaquaticus DNA
polymerasepolymerase
TaqTaq Not permanentlyNot permanently
destroyed at 94ordmCdestroyed at 94ordmC OptimalOptimal
temperature is 72ordmCtemperature is 72ordmC
Problems with TaqProblems with Taq
Taq DNA polymerase - thermostableTaq DNA polymerase - thermostable Lack of 3prime-5prime exonuclease ndash proofreadingLack of 3prime-5prime exonuclease ndash proofreading
Error rate = 2 times 10Error rate = 2 times 10-4-4 nucleotdescycle nucleotdescycle Newer polymerases have high fidelityNewer polymerases have high fidelity
High fidelity polymerase - HiFi TaqHigh fidelity polymerase - HiFi Taq
Termplates for PCRTermplates for PCR
Small amount of templateSmall amount of template In theory a single moleculeIn theory a single molecule Do not need to isolate sequence of interestDo not need to isolate sequence of interest DNA template need not be highly purifiedDNA template need not be highly purified DNA is stable in absence of nucleasesDNA is stable in absence of nucleases
Templates for PCRTemplates for PCR
1048708 1048708 Dried bloodDried blood1048708 1048708 Semen stainsSemen stains1048708 1048708 Vaginal swabsVaginal swabs1048708 1048708 Single hairSingle hair1048708 1048708 Finger nail scrapingsFinger nail scrapings1048708 1048708 Egyptian mummiesEgyptian mummies1048708 1048708 Buccal SwabBuccal Swab1048708 1048708 Tooth brushesTooth brushes
Basic reactionBasic reaction
Thermocycling PCR machine1048707 Previously ndash need to overlay oil to prevent evaporation1048707 Automatically Change temperature1048707 Temperature gradient
The Basics of PCR CyclingThe Basics of PCR Cycling
bull bull 30ndash35 cycles each30ndash35 cycles each
comprisingcomprising
ndash ndash denaturation (95degC)denaturation (95degC)
30 sec30 sec
ndash ndash annealing (55ndash60degC)annealing (55ndash60degC)
30 sec30 sec
ndash ndash extension (72degC)extension (72degC)
time depends ontime depends on
product sizeproduct size
How many copiesHow many copies
bull bull No target products are made until the third No target products are made until the third cyclecycle
bull bull The accumulation is not strictly a doublingThe accumulation is not strictly a doubling
at each cycle in the early phaseat each cycle in the early phase
bull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 target
copies (~1times10copies (~1times1099))
How many cyclesHow many cycles
bull bull Increasing the cycleIncreasing the cyclenumber above ~35 hasnumber above ~35 haslittle positive effectlittle positive effectbull bull The plateau occurs whenThe plateau occurs whenndash ndash The reagents are depletedThe reagents are depletedndash ndash The products re-annealThe products re-annealndash ndash The polymerase isThe polymerase isdamageddamaged- Unwanted products- Unwanted productsaccumulateaccumulate
Basic reactionBasic reaction Oligonucleotide Oligonucleotide
primersprimers Design to flank the Design to flank the
desired sequencedesired sequence Steps include (30-Steps include (30-
40 steps)40 steps) 1048708 1048708 Denaturation at Denaturation at
94degC94degC 1048708 1048708 Primer annealing Primer annealing
at Tm-5degCat Tm-5degC 1048708 1048708 Extension at 72degCExtension at 72degC
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
Amplify DNAAmplify DNAPCRPCR
In vitro amplification (in a test tube)In vitro amplification (in a test tube) Enzymatic Taq polymeraseEnzymatic Taq polymerase
ndash ndash Temperature-resistant DNATemperature-resistant DNA
polymerase ( Thermus aquaticus)polymerase ( Thermus aquaticus) Heat resistantHeat resistant Best for lt2 kb targetBest for lt2 kb target
Takes advantage of basicTakes advantage of basicrequirements of replicationrequirements of replication
A DNA templateA DNA template NucleotidesNucleotides PrimersPrimers polymerasepolymerase
PCR is DNA replication in a test tubePCR is DNA replication in a test tube
PRIMERSPRIMERS
Primers short ssDNA sequencescomplementary to border of sequence of interest
PrimersPrimers
Must have some information Must have some information about sequence flanking your about sequence flanking your targettargetPrimers provide specificityPrimers provide specificity
ends pointing towards each otherends pointing towards each other Complementary to opposite strands with Complementary to opposite strands with
3rsquo3rsquo Should have similar melting temperaturesShould have similar melting temperatures
PrimersPrimers
PCRPCRRegion of interestbetween primers
2 Anneal
3 Extend
Taq polymerase enzymatic extension
PCRRepeated Cycles ofRepeated Cycles of
1 Denaturation 1 Denaturation
2 Annealing2 Annealing
3 Extention 3 Extention
1Denaturation
2 Anneal
3 Extend
Melting temperatureMelting temperature
TTmmooC Temperature at whichC Temperature at which half possible H bonds arehalf possible H bonds are formedformedTTmmooC = 2(A+T) + 4(G+C)C = 2(A+T) + 4(G+C)
55 - -AGACTCAGAGAGAACCC-3AGACTCAGAGAGAACCC-3
4Gs 5Cs 4Gs 5Cs 7As 1T7As 1T
TTmmooC=C= (4x9) + (2x8) = 36+16 = (4x9) + (2x8) = 36+16 = 525200CC
Annealing T =TAnnealing T =Tmm00C -5C -5
Heat-stable polymerase is vitalHeat-stable polymerase is vitalto the ease of the processto the ease of the processhelliphellip
Thermus aquaticusThermus aquaticus
Thermus aquaticus from hot springs inYellowstone National Park USA
The ThermusThe Thermusaquaticus DNAaquaticus DNA
polymerasepolymerase
TaqTaq Not permanentlyNot permanently
destroyed at 94ordmCdestroyed at 94ordmC OptimalOptimal
temperature is 72ordmCtemperature is 72ordmC
Problems with TaqProblems with Taq
Taq DNA polymerase - thermostableTaq DNA polymerase - thermostable Lack of 3prime-5prime exonuclease ndash proofreadingLack of 3prime-5prime exonuclease ndash proofreading
Error rate = 2 times 10Error rate = 2 times 10-4-4 nucleotdescycle nucleotdescycle Newer polymerases have high fidelityNewer polymerases have high fidelity
High fidelity polymerase - HiFi TaqHigh fidelity polymerase - HiFi Taq
Termplates for PCRTermplates for PCR
Small amount of templateSmall amount of template In theory a single moleculeIn theory a single molecule Do not need to isolate sequence of interestDo not need to isolate sequence of interest DNA template need not be highly purifiedDNA template need not be highly purified DNA is stable in absence of nucleasesDNA is stable in absence of nucleases
Templates for PCRTemplates for PCR
1048708 1048708 Dried bloodDried blood1048708 1048708 Semen stainsSemen stains1048708 1048708 Vaginal swabsVaginal swabs1048708 1048708 Single hairSingle hair1048708 1048708 Finger nail scrapingsFinger nail scrapings1048708 1048708 Egyptian mummiesEgyptian mummies1048708 1048708 Buccal SwabBuccal Swab1048708 1048708 Tooth brushesTooth brushes
Basic reactionBasic reaction
Thermocycling PCR machine1048707 Previously ndash need to overlay oil to prevent evaporation1048707 Automatically Change temperature1048707 Temperature gradient
The Basics of PCR CyclingThe Basics of PCR Cycling
bull bull 30ndash35 cycles each30ndash35 cycles each
comprisingcomprising
ndash ndash denaturation (95degC)denaturation (95degC)
30 sec30 sec
ndash ndash annealing (55ndash60degC)annealing (55ndash60degC)
30 sec30 sec
ndash ndash extension (72degC)extension (72degC)
time depends ontime depends on
product sizeproduct size
How many copiesHow many copies
bull bull No target products are made until the third No target products are made until the third cyclecycle
bull bull The accumulation is not strictly a doublingThe accumulation is not strictly a doubling
at each cycle in the early phaseat each cycle in the early phase
bull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 target
copies (~1times10copies (~1times1099))
How many cyclesHow many cycles
bull bull Increasing the cycleIncreasing the cyclenumber above ~35 hasnumber above ~35 haslittle positive effectlittle positive effectbull bull The plateau occurs whenThe plateau occurs whenndash ndash The reagents are depletedThe reagents are depletedndash ndash The products re-annealThe products re-annealndash ndash The polymerase isThe polymerase isdamageddamaged- Unwanted products- Unwanted productsaccumulateaccumulate
Basic reactionBasic reaction Oligonucleotide Oligonucleotide
primersprimers Design to flank the Design to flank the
desired sequencedesired sequence Steps include (30-Steps include (30-
40 steps)40 steps) 1048708 1048708 Denaturation at Denaturation at
94degC94degC 1048708 1048708 Primer annealing Primer annealing
at Tm-5degCat Tm-5degC 1048708 1048708 Extension at 72degCExtension at 72degC
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
Takes advantage of basicTakes advantage of basicrequirements of replicationrequirements of replication
A DNA templateA DNA template NucleotidesNucleotides PrimersPrimers polymerasepolymerase
PCR is DNA replication in a test tubePCR is DNA replication in a test tube
PRIMERSPRIMERS
Primers short ssDNA sequencescomplementary to border of sequence of interest
PrimersPrimers
Must have some information Must have some information about sequence flanking your about sequence flanking your targettargetPrimers provide specificityPrimers provide specificity
ends pointing towards each otherends pointing towards each other Complementary to opposite strands with Complementary to opposite strands with
3rsquo3rsquo Should have similar melting temperaturesShould have similar melting temperatures
PrimersPrimers
PCRPCRRegion of interestbetween primers
2 Anneal
3 Extend
Taq polymerase enzymatic extension
PCRRepeated Cycles ofRepeated Cycles of
1 Denaturation 1 Denaturation
2 Annealing2 Annealing
3 Extention 3 Extention
1Denaturation
2 Anneal
3 Extend
Melting temperatureMelting temperature
TTmmooC Temperature at whichC Temperature at which half possible H bonds arehalf possible H bonds are formedformedTTmmooC = 2(A+T) + 4(G+C)C = 2(A+T) + 4(G+C)
55 - -AGACTCAGAGAGAACCC-3AGACTCAGAGAGAACCC-3
4Gs 5Cs 4Gs 5Cs 7As 1T7As 1T
TTmmooC=C= (4x9) + (2x8) = 36+16 = (4x9) + (2x8) = 36+16 = 525200CC
Annealing T =TAnnealing T =Tmm00C -5C -5
Heat-stable polymerase is vitalHeat-stable polymerase is vitalto the ease of the processto the ease of the processhelliphellip
Thermus aquaticusThermus aquaticus
Thermus aquaticus from hot springs inYellowstone National Park USA
The ThermusThe Thermusaquaticus DNAaquaticus DNA
polymerasepolymerase
TaqTaq Not permanentlyNot permanently
destroyed at 94ordmCdestroyed at 94ordmC OptimalOptimal
temperature is 72ordmCtemperature is 72ordmC
Problems with TaqProblems with Taq
Taq DNA polymerase - thermostableTaq DNA polymerase - thermostable Lack of 3prime-5prime exonuclease ndash proofreadingLack of 3prime-5prime exonuclease ndash proofreading
Error rate = 2 times 10Error rate = 2 times 10-4-4 nucleotdescycle nucleotdescycle Newer polymerases have high fidelityNewer polymerases have high fidelity
High fidelity polymerase - HiFi TaqHigh fidelity polymerase - HiFi Taq
Termplates for PCRTermplates for PCR
Small amount of templateSmall amount of template In theory a single moleculeIn theory a single molecule Do not need to isolate sequence of interestDo not need to isolate sequence of interest DNA template need not be highly purifiedDNA template need not be highly purified DNA is stable in absence of nucleasesDNA is stable in absence of nucleases
Templates for PCRTemplates for PCR
1048708 1048708 Dried bloodDried blood1048708 1048708 Semen stainsSemen stains1048708 1048708 Vaginal swabsVaginal swabs1048708 1048708 Single hairSingle hair1048708 1048708 Finger nail scrapingsFinger nail scrapings1048708 1048708 Egyptian mummiesEgyptian mummies1048708 1048708 Buccal SwabBuccal Swab1048708 1048708 Tooth brushesTooth brushes
Basic reactionBasic reaction
Thermocycling PCR machine1048707 Previously ndash need to overlay oil to prevent evaporation1048707 Automatically Change temperature1048707 Temperature gradient
The Basics of PCR CyclingThe Basics of PCR Cycling
bull bull 30ndash35 cycles each30ndash35 cycles each
comprisingcomprising
ndash ndash denaturation (95degC)denaturation (95degC)
30 sec30 sec
ndash ndash annealing (55ndash60degC)annealing (55ndash60degC)
30 sec30 sec
ndash ndash extension (72degC)extension (72degC)
time depends ontime depends on
product sizeproduct size
How many copiesHow many copies
bull bull No target products are made until the third No target products are made until the third cyclecycle
bull bull The accumulation is not strictly a doublingThe accumulation is not strictly a doubling
at each cycle in the early phaseat each cycle in the early phase
bull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 target
copies (~1times10copies (~1times1099))
How many cyclesHow many cycles
bull bull Increasing the cycleIncreasing the cyclenumber above ~35 hasnumber above ~35 haslittle positive effectlittle positive effectbull bull The plateau occurs whenThe plateau occurs whenndash ndash The reagents are depletedThe reagents are depletedndash ndash The products re-annealThe products re-annealndash ndash The polymerase isThe polymerase isdamageddamaged- Unwanted products- Unwanted productsaccumulateaccumulate
Basic reactionBasic reaction Oligonucleotide Oligonucleotide
primersprimers Design to flank the Design to flank the
desired sequencedesired sequence Steps include (30-Steps include (30-
40 steps)40 steps) 1048708 1048708 Denaturation at Denaturation at
94degC94degC 1048708 1048708 Primer annealing Primer annealing
at Tm-5degCat Tm-5degC 1048708 1048708 Extension at 72degCExtension at 72degC
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
PRIMERSPRIMERS
Primers short ssDNA sequencescomplementary to border of sequence of interest
PrimersPrimers
Must have some information Must have some information about sequence flanking your about sequence flanking your targettargetPrimers provide specificityPrimers provide specificity
ends pointing towards each otherends pointing towards each other Complementary to opposite strands with Complementary to opposite strands with
3rsquo3rsquo Should have similar melting temperaturesShould have similar melting temperatures
PrimersPrimers
PCRPCRRegion of interestbetween primers
2 Anneal
3 Extend
Taq polymerase enzymatic extension
PCRRepeated Cycles ofRepeated Cycles of
1 Denaturation 1 Denaturation
2 Annealing2 Annealing
3 Extention 3 Extention
1Denaturation
2 Anneal
3 Extend
Melting temperatureMelting temperature
TTmmooC Temperature at whichC Temperature at which half possible H bonds arehalf possible H bonds are formedformedTTmmooC = 2(A+T) + 4(G+C)C = 2(A+T) + 4(G+C)
55 - -AGACTCAGAGAGAACCC-3AGACTCAGAGAGAACCC-3
4Gs 5Cs 4Gs 5Cs 7As 1T7As 1T
TTmmooC=C= (4x9) + (2x8) = 36+16 = (4x9) + (2x8) = 36+16 = 525200CC
Annealing T =TAnnealing T =Tmm00C -5C -5
Heat-stable polymerase is vitalHeat-stable polymerase is vitalto the ease of the processto the ease of the processhelliphellip
Thermus aquaticusThermus aquaticus
Thermus aquaticus from hot springs inYellowstone National Park USA
The ThermusThe Thermusaquaticus DNAaquaticus DNA
polymerasepolymerase
TaqTaq Not permanentlyNot permanently
destroyed at 94ordmCdestroyed at 94ordmC OptimalOptimal
temperature is 72ordmCtemperature is 72ordmC
Problems with TaqProblems with Taq
Taq DNA polymerase - thermostableTaq DNA polymerase - thermostable Lack of 3prime-5prime exonuclease ndash proofreadingLack of 3prime-5prime exonuclease ndash proofreading
Error rate = 2 times 10Error rate = 2 times 10-4-4 nucleotdescycle nucleotdescycle Newer polymerases have high fidelityNewer polymerases have high fidelity
High fidelity polymerase - HiFi TaqHigh fidelity polymerase - HiFi Taq
Termplates for PCRTermplates for PCR
Small amount of templateSmall amount of template In theory a single moleculeIn theory a single molecule Do not need to isolate sequence of interestDo not need to isolate sequence of interest DNA template need not be highly purifiedDNA template need not be highly purified DNA is stable in absence of nucleasesDNA is stable in absence of nucleases
Templates for PCRTemplates for PCR
1048708 1048708 Dried bloodDried blood1048708 1048708 Semen stainsSemen stains1048708 1048708 Vaginal swabsVaginal swabs1048708 1048708 Single hairSingle hair1048708 1048708 Finger nail scrapingsFinger nail scrapings1048708 1048708 Egyptian mummiesEgyptian mummies1048708 1048708 Buccal SwabBuccal Swab1048708 1048708 Tooth brushesTooth brushes
Basic reactionBasic reaction
Thermocycling PCR machine1048707 Previously ndash need to overlay oil to prevent evaporation1048707 Automatically Change temperature1048707 Temperature gradient
The Basics of PCR CyclingThe Basics of PCR Cycling
bull bull 30ndash35 cycles each30ndash35 cycles each
comprisingcomprising
ndash ndash denaturation (95degC)denaturation (95degC)
30 sec30 sec
ndash ndash annealing (55ndash60degC)annealing (55ndash60degC)
30 sec30 sec
ndash ndash extension (72degC)extension (72degC)
time depends ontime depends on
product sizeproduct size
How many copiesHow many copies
bull bull No target products are made until the third No target products are made until the third cyclecycle
bull bull The accumulation is not strictly a doublingThe accumulation is not strictly a doubling
at each cycle in the early phaseat each cycle in the early phase
bull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 target
copies (~1times10copies (~1times1099))
How many cyclesHow many cycles
bull bull Increasing the cycleIncreasing the cyclenumber above ~35 hasnumber above ~35 haslittle positive effectlittle positive effectbull bull The plateau occurs whenThe plateau occurs whenndash ndash The reagents are depletedThe reagents are depletedndash ndash The products re-annealThe products re-annealndash ndash The polymerase isThe polymerase isdamageddamaged- Unwanted products- Unwanted productsaccumulateaccumulate
Basic reactionBasic reaction Oligonucleotide Oligonucleotide
primersprimers Design to flank the Design to flank the
desired sequencedesired sequence Steps include (30-Steps include (30-
40 steps)40 steps) 1048708 1048708 Denaturation at Denaturation at
94degC94degC 1048708 1048708 Primer annealing Primer annealing
at Tm-5degCat Tm-5degC 1048708 1048708 Extension at 72degCExtension at 72degC
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
PrimersPrimers
Must have some information Must have some information about sequence flanking your about sequence flanking your targettargetPrimers provide specificityPrimers provide specificity
ends pointing towards each otherends pointing towards each other Complementary to opposite strands with Complementary to opposite strands with
3rsquo3rsquo Should have similar melting temperaturesShould have similar melting temperatures
PrimersPrimers
PCRPCRRegion of interestbetween primers
2 Anneal
3 Extend
Taq polymerase enzymatic extension
PCRRepeated Cycles ofRepeated Cycles of
1 Denaturation 1 Denaturation
2 Annealing2 Annealing
3 Extention 3 Extention
1Denaturation
2 Anneal
3 Extend
Melting temperatureMelting temperature
TTmmooC Temperature at whichC Temperature at which half possible H bonds arehalf possible H bonds are formedformedTTmmooC = 2(A+T) + 4(G+C)C = 2(A+T) + 4(G+C)
55 - -AGACTCAGAGAGAACCC-3AGACTCAGAGAGAACCC-3
4Gs 5Cs 4Gs 5Cs 7As 1T7As 1T
TTmmooC=C= (4x9) + (2x8) = 36+16 = (4x9) + (2x8) = 36+16 = 525200CC
Annealing T =TAnnealing T =Tmm00C -5C -5
Heat-stable polymerase is vitalHeat-stable polymerase is vitalto the ease of the processto the ease of the processhelliphellip
Thermus aquaticusThermus aquaticus
Thermus aquaticus from hot springs inYellowstone National Park USA
The ThermusThe Thermusaquaticus DNAaquaticus DNA
polymerasepolymerase
TaqTaq Not permanentlyNot permanently
destroyed at 94ordmCdestroyed at 94ordmC OptimalOptimal
temperature is 72ordmCtemperature is 72ordmC
Problems with TaqProblems with Taq
Taq DNA polymerase - thermostableTaq DNA polymerase - thermostable Lack of 3prime-5prime exonuclease ndash proofreadingLack of 3prime-5prime exonuclease ndash proofreading
Error rate = 2 times 10Error rate = 2 times 10-4-4 nucleotdescycle nucleotdescycle Newer polymerases have high fidelityNewer polymerases have high fidelity
High fidelity polymerase - HiFi TaqHigh fidelity polymerase - HiFi Taq
Termplates for PCRTermplates for PCR
Small amount of templateSmall amount of template In theory a single moleculeIn theory a single molecule Do not need to isolate sequence of interestDo not need to isolate sequence of interest DNA template need not be highly purifiedDNA template need not be highly purified DNA is stable in absence of nucleasesDNA is stable in absence of nucleases
Templates for PCRTemplates for PCR
1048708 1048708 Dried bloodDried blood1048708 1048708 Semen stainsSemen stains1048708 1048708 Vaginal swabsVaginal swabs1048708 1048708 Single hairSingle hair1048708 1048708 Finger nail scrapingsFinger nail scrapings1048708 1048708 Egyptian mummiesEgyptian mummies1048708 1048708 Buccal SwabBuccal Swab1048708 1048708 Tooth brushesTooth brushes
Basic reactionBasic reaction
Thermocycling PCR machine1048707 Previously ndash need to overlay oil to prevent evaporation1048707 Automatically Change temperature1048707 Temperature gradient
The Basics of PCR CyclingThe Basics of PCR Cycling
bull bull 30ndash35 cycles each30ndash35 cycles each
comprisingcomprising
ndash ndash denaturation (95degC)denaturation (95degC)
30 sec30 sec
ndash ndash annealing (55ndash60degC)annealing (55ndash60degC)
30 sec30 sec
ndash ndash extension (72degC)extension (72degC)
time depends ontime depends on
product sizeproduct size
How many copiesHow many copies
bull bull No target products are made until the third No target products are made until the third cyclecycle
bull bull The accumulation is not strictly a doublingThe accumulation is not strictly a doubling
at each cycle in the early phaseat each cycle in the early phase
bull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 target
copies (~1times10copies (~1times1099))
How many cyclesHow many cycles
bull bull Increasing the cycleIncreasing the cyclenumber above ~35 hasnumber above ~35 haslittle positive effectlittle positive effectbull bull The plateau occurs whenThe plateau occurs whenndash ndash The reagents are depletedThe reagents are depletedndash ndash The products re-annealThe products re-annealndash ndash The polymerase isThe polymerase isdamageddamaged- Unwanted products- Unwanted productsaccumulateaccumulate
Basic reactionBasic reaction Oligonucleotide Oligonucleotide
primersprimers Design to flank the Design to flank the
desired sequencedesired sequence Steps include (30-Steps include (30-
40 steps)40 steps) 1048708 1048708 Denaturation at Denaturation at
94degC94degC 1048708 1048708 Primer annealing Primer annealing
at Tm-5degCat Tm-5degC 1048708 1048708 Extension at 72degCExtension at 72degC
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
ends pointing towards each otherends pointing towards each other Complementary to opposite strands with Complementary to opposite strands with
3rsquo3rsquo Should have similar melting temperaturesShould have similar melting temperatures
PrimersPrimers
PCRPCRRegion of interestbetween primers
2 Anneal
3 Extend
Taq polymerase enzymatic extension
PCRRepeated Cycles ofRepeated Cycles of
1 Denaturation 1 Denaturation
2 Annealing2 Annealing
3 Extention 3 Extention
1Denaturation
2 Anneal
3 Extend
Melting temperatureMelting temperature
TTmmooC Temperature at whichC Temperature at which half possible H bonds arehalf possible H bonds are formedformedTTmmooC = 2(A+T) + 4(G+C)C = 2(A+T) + 4(G+C)
55 - -AGACTCAGAGAGAACCC-3AGACTCAGAGAGAACCC-3
4Gs 5Cs 4Gs 5Cs 7As 1T7As 1T
TTmmooC=C= (4x9) + (2x8) = 36+16 = (4x9) + (2x8) = 36+16 = 525200CC
Annealing T =TAnnealing T =Tmm00C -5C -5
Heat-stable polymerase is vitalHeat-stable polymerase is vitalto the ease of the processto the ease of the processhelliphellip
Thermus aquaticusThermus aquaticus
Thermus aquaticus from hot springs inYellowstone National Park USA
The ThermusThe Thermusaquaticus DNAaquaticus DNA
polymerasepolymerase
TaqTaq Not permanentlyNot permanently
destroyed at 94ordmCdestroyed at 94ordmC OptimalOptimal
temperature is 72ordmCtemperature is 72ordmC
Problems with TaqProblems with Taq
Taq DNA polymerase - thermostableTaq DNA polymerase - thermostable Lack of 3prime-5prime exonuclease ndash proofreadingLack of 3prime-5prime exonuclease ndash proofreading
Error rate = 2 times 10Error rate = 2 times 10-4-4 nucleotdescycle nucleotdescycle Newer polymerases have high fidelityNewer polymerases have high fidelity
High fidelity polymerase - HiFi TaqHigh fidelity polymerase - HiFi Taq
Termplates for PCRTermplates for PCR
Small amount of templateSmall amount of template In theory a single moleculeIn theory a single molecule Do not need to isolate sequence of interestDo not need to isolate sequence of interest DNA template need not be highly purifiedDNA template need not be highly purified DNA is stable in absence of nucleasesDNA is stable in absence of nucleases
Templates for PCRTemplates for PCR
1048708 1048708 Dried bloodDried blood1048708 1048708 Semen stainsSemen stains1048708 1048708 Vaginal swabsVaginal swabs1048708 1048708 Single hairSingle hair1048708 1048708 Finger nail scrapingsFinger nail scrapings1048708 1048708 Egyptian mummiesEgyptian mummies1048708 1048708 Buccal SwabBuccal Swab1048708 1048708 Tooth brushesTooth brushes
Basic reactionBasic reaction
Thermocycling PCR machine1048707 Previously ndash need to overlay oil to prevent evaporation1048707 Automatically Change temperature1048707 Temperature gradient
The Basics of PCR CyclingThe Basics of PCR Cycling
bull bull 30ndash35 cycles each30ndash35 cycles each
comprisingcomprising
ndash ndash denaturation (95degC)denaturation (95degC)
30 sec30 sec
ndash ndash annealing (55ndash60degC)annealing (55ndash60degC)
30 sec30 sec
ndash ndash extension (72degC)extension (72degC)
time depends ontime depends on
product sizeproduct size
How many copiesHow many copies
bull bull No target products are made until the third No target products are made until the third cyclecycle
bull bull The accumulation is not strictly a doublingThe accumulation is not strictly a doubling
at each cycle in the early phaseat each cycle in the early phase
bull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 target
copies (~1times10copies (~1times1099))
How many cyclesHow many cycles
bull bull Increasing the cycleIncreasing the cyclenumber above ~35 hasnumber above ~35 haslittle positive effectlittle positive effectbull bull The plateau occurs whenThe plateau occurs whenndash ndash The reagents are depletedThe reagents are depletedndash ndash The products re-annealThe products re-annealndash ndash The polymerase isThe polymerase isdamageddamaged- Unwanted products- Unwanted productsaccumulateaccumulate
Basic reactionBasic reaction Oligonucleotide Oligonucleotide
primersprimers Design to flank the Design to flank the
desired sequencedesired sequence Steps include (30-Steps include (30-
40 steps)40 steps) 1048708 1048708 Denaturation at Denaturation at
94degC94degC 1048708 1048708 Primer annealing Primer annealing
at Tm-5degCat Tm-5degC 1048708 1048708 Extension at 72degCExtension at 72degC
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
PCRPCRRegion of interestbetween primers
2 Anneal
3 Extend
Taq polymerase enzymatic extension
PCRRepeated Cycles ofRepeated Cycles of
1 Denaturation 1 Denaturation
2 Annealing2 Annealing
3 Extention 3 Extention
1Denaturation
2 Anneal
3 Extend
Melting temperatureMelting temperature
TTmmooC Temperature at whichC Temperature at which half possible H bonds arehalf possible H bonds are formedformedTTmmooC = 2(A+T) + 4(G+C)C = 2(A+T) + 4(G+C)
55 - -AGACTCAGAGAGAACCC-3AGACTCAGAGAGAACCC-3
4Gs 5Cs 4Gs 5Cs 7As 1T7As 1T
TTmmooC=C= (4x9) + (2x8) = 36+16 = (4x9) + (2x8) = 36+16 = 525200CC
Annealing T =TAnnealing T =Tmm00C -5C -5
Heat-stable polymerase is vitalHeat-stable polymerase is vitalto the ease of the processto the ease of the processhelliphellip
Thermus aquaticusThermus aquaticus
Thermus aquaticus from hot springs inYellowstone National Park USA
The ThermusThe Thermusaquaticus DNAaquaticus DNA
polymerasepolymerase
TaqTaq Not permanentlyNot permanently
destroyed at 94ordmCdestroyed at 94ordmC OptimalOptimal
temperature is 72ordmCtemperature is 72ordmC
Problems with TaqProblems with Taq
Taq DNA polymerase - thermostableTaq DNA polymerase - thermostable Lack of 3prime-5prime exonuclease ndash proofreadingLack of 3prime-5prime exonuclease ndash proofreading
Error rate = 2 times 10Error rate = 2 times 10-4-4 nucleotdescycle nucleotdescycle Newer polymerases have high fidelityNewer polymerases have high fidelity
High fidelity polymerase - HiFi TaqHigh fidelity polymerase - HiFi Taq
Termplates for PCRTermplates for PCR
Small amount of templateSmall amount of template In theory a single moleculeIn theory a single molecule Do not need to isolate sequence of interestDo not need to isolate sequence of interest DNA template need not be highly purifiedDNA template need not be highly purified DNA is stable in absence of nucleasesDNA is stable in absence of nucleases
Templates for PCRTemplates for PCR
1048708 1048708 Dried bloodDried blood1048708 1048708 Semen stainsSemen stains1048708 1048708 Vaginal swabsVaginal swabs1048708 1048708 Single hairSingle hair1048708 1048708 Finger nail scrapingsFinger nail scrapings1048708 1048708 Egyptian mummiesEgyptian mummies1048708 1048708 Buccal SwabBuccal Swab1048708 1048708 Tooth brushesTooth brushes
Basic reactionBasic reaction
Thermocycling PCR machine1048707 Previously ndash need to overlay oil to prevent evaporation1048707 Automatically Change temperature1048707 Temperature gradient
The Basics of PCR CyclingThe Basics of PCR Cycling
bull bull 30ndash35 cycles each30ndash35 cycles each
comprisingcomprising
ndash ndash denaturation (95degC)denaturation (95degC)
30 sec30 sec
ndash ndash annealing (55ndash60degC)annealing (55ndash60degC)
30 sec30 sec
ndash ndash extension (72degC)extension (72degC)
time depends ontime depends on
product sizeproduct size
How many copiesHow many copies
bull bull No target products are made until the third No target products are made until the third cyclecycle
bull bull The accumulation is not strictly a doublingThe accumulation is not strictly a doubling
at each cycle in the early phaseat each cycle in the early phase
bull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 target
copies (~1times10copies (~1times1099))
How many cyclesHow many cycles
bull bull Increasing the cycleIncreasing the cyclenumber above ~35 hasnumber above ~35 haslittle positive effectlittle positive effectbull bull The plateau occurs whenThe plateau occurs whenndash ndash The reagents are depletedThe reagents are depletedndash ndash The products re-annealThe products re-annealndash ndash The polymerase isThe polymerase isdamageddamaged- Unwanted products- Unwanted productsaccumulateaccumulate
Basic reactionBasic reaction Oligonucleotide Oligonucleotide
primersprimers Design to flank the Design to flank the
desired sequencedesired sequence Steps include (30-Steps include (30-
40 steps)40 steps) 1048708 1048708 Denaturation at Denaturation at
94degC94degC 1048708 1048708 Primer annealing Primer annealing
at Tm-5degCat Tm-5degC 1048708 1048708 Extension at 72degCExtension at 72degC
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
PCRRepeated Cycles ofRepeated Cycles of
1 Denaturation 1 Denaturation
2 Annealing2 Annealing
3 Extention 3 Extention
1Denaturation
2 Anneal
3 Extend
Melting temperatureMelting temperature
TTmmooC Temperature at whichC Temperature at which half possible H bonds arehalf possible H bonds are formedformedTTmmooC = 2(A+T) + 4(G+C)C = 2(A+T) + 4(G+C)
55 - -AGACTCAGAGAGAACCC-3AGACTCAGAGAGAACCC-3
4Gs 5Cs 4Gs 5Cs 7As 1T7As 1T
TTmmooC=C= (4x9) + (2x8) = 36+16 = (4x9) + (2x8) = 36+16 = 525200CC
Annealing T =TAnnealing T =Tmm00C -5C -5
Heat-stable polymerase is vitalHeat-stable polymerase is vitalto the ease of the processto the ease of the processhelliphellip
Thermus aquaticusThermus aquaticus
Thermus aquaticus from hot springs inYellowstone National Park USA
The ThermusThe Thermusaquaticus DNAaquaticus DNA
polymerasepolymerase
TaqTaq Not permanentlyNot permanently
destroyed at 94ordmCdestroyed at 94ordmC OptimalOptimal
temperature is 72ordmCtemperature is 72ordmC
Problems with TaqProblems with Taq
Taq DNA polymerase - thermostableTaq DNA polymerase - thermostable Lack of 3prime-5prime exonuclease ndash proofreadingLack of 3prime-5prime exonuclease ndash proofreading
Error rate = 2 times 10Error rate = 2 times 10-4-4 nucleotdescycle nucleotdescycle Newer polymerases have high fidelityNewer polymerases have high fidelity
High fidelity polymerase - HiFi TaqHigh fidelity polymerase - HiFi Taq
Termplates for PCRTermplates for PCR
Small amount of templateSmall amount of template In theory a single moleculeIn theory a single molecule Do not need to isolate sequence of interestDo not need to isolate sequence of interest DNA template need not be highly purifiedDNA template need not be highly purified DNA is stable in absence of nucleasesDNA is stable in absence of nucleases
Templates for PCRTemplates for PCR
1048708 1048708 Dried bloodDried blood1048708 1048708 Semen stainsSemen stains1048708 1048708 Vaginal swabsVaginal swabs1048708 1048708 Single hairSingle hair1048708 1048708 Finger nail scrapingsFinger nail scrapings1048708 1048708 Egyptian mummiesEgyptian mummies1048708 1048708 Buccal SwabBuccal Swab1048708 1048708 Tooth brushesTooth brushes
Basic reactionBasic reaction
Thermocycling PCR machine1048707 Previously ndash need to overlay oil to prevent evaporation1048707 Automatically Change temperature1048707 Temperature gradient
The Basics of PCR CyclingThe Basics of PCR Cycling
bull bull 30ndash35 cycles each30ndash35 cycles each
comprisingcomprising
ndash ndash denaturation (95degC)denaturation (95degC)
30 sec30 sec
ndash ndash annealing (55ndash60degC)annealing (55ndash60degC)
30 sec30 sec
ndash ndash extension (72degC)extension (72degC)
time depends ontime depends on
product sizeproduct size
How many copiesHow many copies
bull bull No target products are made until the third No target products are made until the third cyclecycle
bull bull The accumulation is not strictly a doublingThe accumulation is not strictly a doubling
at each cycle in the early phaseat each cycle in the early phase
bull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 target
copies (~1times10copies (~1times1099))
How many cyclesHow many cycles
bull bull Increasing the cycleIncreasing the cyclenumber above ~35 hasnumber above ~35 haslittle positive effectlittle positive effectbull bull The plateau occurs whenThe plateau occurs whenndash ndash The reagents are depletedThe reagents are depletedndash ndash The products re-annealThe products re-annealndash ndash The polymerase isThe polymerase isdamageddamaged- Unwanted products- Unwanted productsaccumulateaccumulate
Basic reactionBasic reaction Oligonucleotide Oligonucleotide
primersprimers Design to flank the Design to flank the
desired sequencedesired sequence Steps include (30-Steps include (30-
40 steps)40 steps) 1048708 1048708 Denaturation at Denaturation at
94degC94degC 1048708 1048708 Primer annealing Primer annealing
at Tm-5degCat Tm-5degC 1048708 1048708 Extension at 72degCExtension at 72degC
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
Melting temperatureMelting temperature
TTmmooC Temperature at whichC Temperature at which half possible H bonds arehalf possible H bonds are formedformedTTmmooC = 2(A+T) + 4(G+C)C = 2(A+T) + 4(G+C)
55 - -AGACTCAGAGAGAACCC-3AGACTCAGAGAGAACCC-3
4Gs 5Cs 4Gs 5Cs 7As 1T7As 1T
TTmmooC=C= (4x9) + (2x8) = 36+16 = (4x9) + (2x8) = 36+16 = 525200CC
Annealing T =TAnnealing T =Tmm00C -5C -5
Heat-stable polymerase is vitalHeat-stable polymerase is vitalto the ease of the processto the ease of the processhelliphellip
Thermus aquaticusThermus aquaticus
Thermus aquaticus from hot springs inYellowstone National Park USA
The ThermusThe Thermusaquaticus DNAaquaticus DNA
polymerasepolymerase
TaqTaq Not permanentlyNot permanently
destroyed at 94ordmCdestroyed at 94ordmC OptimalOptimal
temperature is 72ordmCtemperature is 72ordmC
Problems with TaqProblems with Taq
Taq DNA polymerase - thermostableTaq DNA polymerase - thermostable Lack of 3prime-5prime exonuclease ndash proofreadingLack of 3prime-5prime exonuclease ndash proofreading
Error rate = 2 times 10Error rate = 2 times 10-4-4 nucleotdescycle nucleotdescycle Newer polymerases have high fidelityNewer polymerases have high fidelity
High fidelity polymerase - HiFi TaqHigh fidelity polymerase - HiFi Taq
Termplates for PCRTermplates for PCR
Small amount of templateSmall amount of template In theory a single moleculeIn theory a single molecule Do not need to isolate sequence of interestDo not need to isolate sequence of interest DNA template need not be highly purifiedDNA template need not be highly purified DNA is stable in absence of nucleasesDNA is stable in absence of nucleases
Templates for PCRTemplates for PCR
1048708 1048708 Dried bloodDried blood1048708 1048708 Semen stainsSemen stains1048708 1048708 Vaginal swabsVaginal swabs1048708 1048708 Single hairSingle hair1048708 1048708 Finger nail scrapingsFinger nail scrapings1048708 1048708 Egyptian mummiesEgyptian mummies1048708 1048708 Buccal SwabBuccal Swab1048708 1048708 Tooth brushesTooth brushes
Basic reactionBasic reaction
Thermocycling PCR machine1048707 Previously ndash need to overlay oil to prevent evaporation1048707 Automatically Change temperature1048707 Temperature gradient
The Basics of PCR CyclingThe Basics of PCR Cycling
bull bull 30ndash35 cycles each30ndash35 cycles each
comprisingcomprising
ndash ndash denaturation (95degC)denaturation (95degC)
30 sec30 sec
ndash ndash annealing (55ndash60degC)annealing (55ndash60degC)
30 sec30 sec
ndash ndash extension (72degC)extension (72degC)
time depends ontime depends on
product sizeproduct size
How many copiesHow many copies
bull bull No target products are made until the third No target products are made until the third cyclecycle
bull bull The accumulation is not strictly a doublingThe accumulation is not strictly a doubling
at each cycle in the early phaseat each cycle in the early phase
bull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 target
copies (~1times10copies (~1times1099))
How many cyclesHow many cycles
bull bull Increasing the cycleIncreasing the cyclenumber above ~35 hasnumber above ~35 haslittle positive effectlittle positive effectbull bull The plateau occurs whenThe plateau occurs whenndash ndash The reagents are depletedThe reagents are depletedndash ndash The products re-annealThe products re-annealndash ndash The polymerase isThe polymerase isdamageddamaged- Unwanted products- Unwanted productsaccumulateaccumulate
Basic reactionBasic reaction Oligonucleotide Oligonucleotide
primersprimers Design to flank the Design to flank the
desired sequencedesired sequence Steps include (30-Steps include (30-
40 steps)40 steps) 1048708 1048708 Denaturation at Denaturation at
94degC94degC 1048708 1048708 Primer annealing Primer annealing
at Tm-5degCat Tm-5degC 1048708 1048708 Extension at 72degCExtension at 72degC
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
Heat-stable polymerase is vitalHeat-stable polymerase is vitalto the ease of the processto the ease of the processhelliphellip
Thermus aquaticusThermus aquaticus
Thermus aquaticus from hot springs inYellowstone National Park USA
The ThermusThe Thermusaquaticus DNAaquaticus DNA
polymerasepolymerase
TaqTaq Not permanentlyNot permanently
destroyed at 94ordmCdestroyed at 94ordmC OptimalOptimal
temperature is 72ordmCtemperature is 72ordmC
Problems with TaqProblems with Taq
Taq DNA polymerase - thermostableTaq DNA polymerase - thermostable Lack of 3prime-5prime exonuclease ndash proofreadingLack of 3prime-5prime exonuclease ndash proofreading
Error rate = 2 times 10Error rate = 2 times 10-4-4 nucleotdescycle nucleotdescycle Newer polymerases have high fidelityNewer polymerases have high fidelity
High fidelity polymerase - HiFi TaqHigh fidelity polymerase - HiFi Taq
Termplates for PCRTermplates for PCR
Small amount of templateSmall amount of template In theory a single moleculeIn theory a single molecule Do not need to isolate sequence of interestDo not need to isolate sequence of interest DNA template need not be highly purifiedDNA template need not be highly purified DNA is stable in absence of nucleasesDNA is stable in absence of nucleases
Templates for PCRTemplates for PCR
1048708 1048708 Dried bloodDried blood1048708 1048708 Semen stainsSemen stains1048708 1048708 Vaginal swabsVaginal swabs1048708 1048708 Single hairSingle hair1048708 1048708 Finger nail scrapingsFinger nail scrapings1048708 1048708 Egyptian mummiesEgyptian mummies1048708 1048708 Buccal SwabBuccal Swab1048708 1048708 Tooth brushesTooth brushes
Basic reactionBasic reaction
Thermocycling PCR machine1048707 Previously ndash need to overlay oil to prevent evaporation1048707 Automatically Change temperature1048707 Temperature gradient
The Basics of PCR CyclingThe Basics of PCR Cycling
bull bull 30ndash35 cycles each30ndash35 cycles each
comprisingcomprising
ndash ndash denaturation (95degC)denaturation (95degC)
30 sec30 sec
ndash ndash annealing (55ndash60degC)annealing (55ndash60degC)
30 sec30 sec
ndash ndash extension (72degC)extension (72degC)
time depends ontime depends on
product sizeproduct size
How many copiesHow many copies
bull bull No target products are made until the third No target products are made until the third cyclecycle
bull bull The accumulation is not strictly a doublingThe accumulation is not strictly a doubling
at each cycle in the early phaseat each cycle in the early phase
bull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 target
copies (~1times10copies (~1times1099))
How many cyclesHow many cycles
bull bull Increasing the cycleIncreasing the cyclenumber above ~35 hasnumber above ~35 haslittle positive effectlittle positive effectbull bull The plateau occurs whenThe plateau occurs whenndash ndash The reagents are depletedThe reagents are depletedndash ndash The products re-annealThe products re-annealndash ndash The polymerase isThe polymerase isdamageddamaged- Unwanted products- Unwanted productsaccumulateaccumulate
Basic reactionBasic reaction Oligonucleotide Oligonucleotide
primersprimers Design to flank the Design to flank the
desired sequencedesired sequence Steps include (30-Steps include (30-
40 steps)40 steps) 1048708 1048708 Denaturation at Denaturation at
94degC94degC 1048708 1048708 Primer annealing Primer annealing
at Tm-5degCat Tm-5degC 1048708 1048708 Extension at 72degCExtension at 72degC
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
Thermus aquaticusThermus aquaticus
Thermus aquaticus from hot springs inYellowstone National Park USA
The ThermusThe Thermusaquaticus DNAaquaticus DNA
polymerasepolymerase
TaqTaq Not permanentlyNot permanently
destroyed at 94ordmCdestroyed at 94ordmC OptimalOptimal
temperature is 72ordmCtemperature is 72ordmC
Problems with TaqProblems with Taq
Taq DNA polymerase - thermostableTaq DNA polymerase - thermostable Lack of 3prime-5prime exonuclease ndash proofreadingLack of 3prime-5prime exonuclease ndash proofreading
Error rate = 2 times 10Error rate = 2 times 10-4-4 nucleotdescycle nucleotdescycle Newer polymerases have high fidelityNewer polymerases have high fidelity
High fidelity polymerase - HiFi TaqHigh fidelity polymerase - HiFi Taq
Termplates for PCRTermplates for PCR
Small amount of templateSmall amount of template In theory a single moleculeIn theory a single molecule Do not need to isolate sequence of interestDo not need to isolate sequence of interest DNA template need not be highly purifiedDNA template need not be highly purified DNA is stable in absence of nucleasesDNA is stable in absence of nucleases
Templates for PCRTemplates for PCR
1048708 1048708 Dried bloodDried blood1048708 1048708 Semen stainsSemen stains1048708 1048708 Vaginal swabsVaginal swabs1048708 1048708 Single hairSingle hair1048708 1048708 Finger nail scrapingsFinger nail scrapings1048708 1048708 Egyptian mummiesEgyptian mummies1048708 1048708 Buccal SwabBuccal Swab1048708 1048708 Tooth brushesTooth brushes
Basic reactionBasic reaction
Thermocycling PCR machine1048707 Previously ndash need to overlay oil to prevent evaporation1048707 Automatically Change temperature1048707 Temperature gradient
The Basics of PCR CyclingThe Basics of PCR Cycling
bull bull 30ndash35 cycles each30ndash35 cycles each
comprisingcomprising
ndash ndash denaturation (95degC)denaturation (95degC)
30 sec30 sec
ndash ndash annealing (55ndash60degC)annealing (55ndash60degC)
30 sec30 sec
ndash ndash extension (72degC)extension (72degC)
time depends ontime depends on
product sizeproduct size
How many copiesHow many copies
bull bull No target products are made until the third No target products are made until the third cyclecycle
bull bull The accumulation is not strictly a doublingThe accumulation is not strictly a doubling
at each cycle in the early phaseat each cycle in the early phase
bull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 target
copies (~1times10copies (~1times1099))
How many cyclesHow many cycles
bull bull Increasing the cycleIncreasing the cyclenumber above ~35 hasnumber above ~35 haslittle positive effectlittle positive effectbull bull The plateau occurs whenThe plateau occurs whenndash ndash The reagents are depletedThe reagents are depletedndash ndash The products re-annealThe products re-annealndash ndash The polymerase isThe polymerase isdamageddamaged- Unwanted products- Unwanted productsaccumulateaccumulate
Basic reactionBasic reaction Oligonucleotide Oligonucleotide
primersprimers Design to flank the Design to flank the
desired sequencedesired sequence Steps include (30-Steps include (30-
40 steps)40 steps) 1048708 1048708 Denaturation at Denaturation at
94degC94degC 1048708 1048708 Primer annealing Primer annealing
at Tm-5degCat Tm-5degC 1048708 1048708 Extension at 72degCExtension at 72degC
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
The ThermusThe Thermusaquaticus DNAaquaticus DNA
polymerasepolymerase
TaqTaq Not permanentlyNot permanently
destroyed at 94ordmCdestroyed at 94ordmC OptimalOptimal
temperature is 72ordmCtemperature is 72ordmC
Problems with TaqProblems with Taq
Taq DNA polymerase - thermostableTaq DNA polymerase - thermostable Lack of 3prime-5prime exonuclease ndash proofreadingLack of 3prime-5prime exonuclease ndash proofreading
Error rate = 2 times 10Error rate = 2 times 10-4-4 nucleotdescycle nucleotdescycle Newer polymerases have high fidelityNewer polymerases have high fidelity
High fidelity polymerase - HiFi TaqHigh fidelity polymerase - HiFi Taq
Termplates for PCRTermplates for PCR
Small amount of templateSmall amount of template In theory a single moleculeIn theory a single molecule Do not need to isolate sequence of interestDo not need to isolate sequence of interest DNA template need not be highly purifiedDNA template need not be highly purified DNA is stable in absence of nucleasesDNA is stable in absence of nucleases
Templates for PCRTemplates for PCR
1048708 1048708 Dried bloodDried blood1048708 1048708 Semen stainsSemen stains1048708 1048708 Vaginal swabsVaginal swabs1048708 1048708 Single hairSingle hair1048708 1048708 Finger nail scrapingsFinger nail scrapings1048708 1048708 Egyptian mummiesEgyptian mummies1048708 1048708 Buccal SwabBuccal Swab1048708 1048708 Tooth brushesTooth brushes
Basic reactionBasic reaction
Thermocycling PCR machine1048707 Previously ndash need to overlay oil to prevent evaporation1048707 Automatically Change temperature1048707 Temperature gradient
The Basics of PCR CyclingThe Basics of PCR Cycling
bull bull 30ndash35 cycles each30ndash35 cycles each
comprisingcomprising
ndash ndash denaturation (95degC)denaturation (95degC)
30 sec30 sec
ndash ndash annealing (55ndash60degC)annealing (55ndash60degC)
30 sec30 sec
ndash ndash extension (72degC)extension (72degC)
time depends ontime depends on
product sizeproduct size
How many copiesHow many copies
bull bull No target products are made until the third No target products are made until the third cyclecycle
bull bull The accumulation is not strictly a doublingThe accumulation is not strictly a doubling
at each cycle in the early phaseat each cycle in the early phase
bull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 target
copies (~1times10copies (~1times1099))
How many cyclesHow many cycles
bull bull Increasing the cycleIncreasing the cyclenumber above ~35 hasnumber above ~35 haslittle positive effectlittle positive effectbull bull The plateau occurs whenThe plateau occurs whenndash ndash The reagents are depletedThe reagents are depletedndash ndash The products re-annealThe products re-annealndash ndash The polymerase isThe polymerase isdamageddamaged- Unwanted products- Unwanted productsaccumulateaccumulate
Basic reactionBasic reaction Oligonucleotide Oligonucleotide
primersprimers Design to flank the Design to flank the
desired sequencedesired sequence Steps include (30-Steps include (30-
40 steps)40 steps) 1048708 1048708 Denaturation at Denaturation at
94degC94degC 1048708 1048708 Primer annealing Primer annealing
at Tm-5degCat Tm-5degC 1048708 1048708 Extension at 72degCExtension at 72degC
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
Problems with TaqProblems with Taq
Taq DNA polymerase - thermostableTaq DNA polymerase - thermostable Lack of 3prime-5prime exonuclease ndash proofreadingLack of 3prime-5prime exonuclease ndash proofreading
Error rate = 2 times 10Error rate = 2 times 10-4-4 nucleotdescycle nucleotdescycle Newer polymerases have high fidelityNewer polymerases have high fidelity
High fidelity polymerase - HiFi TaqHigh fidelity polymerase - HiFi Taq
Termplates for PCRTermplates for PCR
Small amount of templateSmall amount of template In theory a single moleculeIn theory a single molecule Do not need to isolate sequence of interestDo not need to isolate sequence of interest DNA template need not be highly purifiedDNA template need not be highly purified DNA is stable in absence of nucleasesDNA is stable in absence of nucleases
Templates for PCRTemplates for PCR
1048708 1048708 Dried bloodDried blood1048708 1048708 Semen stainsSemen stains1048708 1048708 Vaginal swabsVaginal swabs1048708 1048708 Single hairSingle hair1048708 1048708 Finger nail scrapingsFinger nail scrapings1048708 1048708 Egyptian mummiesEgyptian mummies1048708 1048708 Buccal SwabBuccal Swab1048708 1048708 Tooth brushesTooth brushes
Basic reactionBasic reaction
Thermocycling PCR machine1048707 Previously ndash need to overlay oil to prevent evaporation1048707 Automatically Change temperature1048707 Temperature gradient
The Basics of PCR CyclingThe Basics of PCR Cycling
bull bull 30ndash35 cycles each30ndash35 cycles each
comprisingcomprising
ndash ndash denaturation (95degC)denaturation (95degC)
30 sec30 sec
ndash ndash annealing (55ndash60degC)annealing (55ndash60degC)
30 sec30 sec
ndash ndash extension (72degC)extension (72degC)
time depends ontime depends on
product sizeproduct size
How many copiesHow many copies
bull bull No target products are made until the third No target products are made until the third cyclecycle
bull bull The accumulation is not strictly a doublingThe accumulation is not strictly a doubling
at each cycle in the early phaseat each cycle in the early phase
bull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 target
copies (~1times10copies (~1times1099))
How many cyclesHow many cycles
bull bull Increasing the cycleIncreasing the cyclenumber above ~35 hasnumber above ~35 haslittle positive effectlittle positive effectbull bull The plateau occurs whenThe plateau occurs whenndash ndash The reagents are depletedThe reagents are depletedndash ndash The products re-annealThe products re-annealndash ndash The polymerase isThe polymerase isdamageddamaged- Unwanted products- Unwanted productsaccumulateaccumulate
Basic reactionBasic reaction Oligonucleotide Oligonucleotide
primersprimers Design to flank the Design to flank the
desired sequencedesired sequence Steps include (30-Steps include (30-
40 steps)40 steps) 1048708 1048708 Denaturation at Denaturation at
94degC94degC 1048708 1048708 Primer annealing Primer annealing
at Tm-5degCat Tm-5degC 1048708 1048708 Extension at 72degCExtension at 72degC
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
Termplates for PCRTermplates for PCR
Small amount of templateSmall amount of template In theory a single moleculeIn theory a single molecule Do not need to isolate sequence of interestDo not need to isolate sequence of interest DNA template need not be highly purifiedDNA template need not be highly purified DNA is stable in absence of nucleasesDNA is stable in absence of nucleases
Templates for PCRTemplates for PCR
1048708 1048708 Dried bloodDried blood1048708 1048708 Semen stainsSemen stains1048708 1048708 Vaginal swabsVaginal swabs1048708 1048708 Single hairSingle hair1048708 1048708 Finger nail scrapingsFinger nail scrapings1048708 1048708 Egyptian mummiesEgyptian mummies1048708 1048708 Buccal SwabBuccal Swab1048708 1048708 Tooth brushesTooth brushes
Basic reactionBasic reaction
Thermocycling PCR machine1048707 Previously ndash need to overlay oil to prevent evaporation1048707 Automatically Change temperature1048707 Temperature gradient
The Basics of PCR CyclingThe Basics of PCR Cycling
bull bull 30ndash35 cycles each30ndash35 cycles each
comprisingcomprising
ndash ndash denaturation (95degC)denaturation (95degC)
30 sec30 sec
ndash ndash annealing (55ndash60degC)annealing (55ndash60degC)
30 sec30 sec
ndash ndash extension (72degC)extension (72degC)
time depends ontime depends on
product sizeproduct size
How many copiesHow many copies
bull bull No target products are made until the third No target products are made until the third cyclecycle
bull bull The accumulation is not strictly a doublingThe accumulation is not strictly a doubling
at each cycle in the early phaseat each cycle in the early phase
bull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 target
copies (~1times10copies (~1times1099))
How many cyclesHow many cycles
bull bull Increasing the cycleIncreasing the cyclenumber above ~35 hasnumber above ~35 haslittle positive effectlittle positive effectbull bull The plateau occurs whenThe plateau occurs whenndash ndash The reagents are depletedThe reagents are depletedndash ndash The products re-annealThe products re-annealndash ndash The polymerase isThe polymerase isdamageddamaged- Unwanted products- Unwanted productsaccumulateaccumulate
Basic reactionBasic reaction Oligonucleotide Oligonucleotide
primersprimers Design to flank the Design to flank the
desired sequencedesired sequence Steps include (30-Steps include (30-
40 steps)40 steps) 1048708 1048708 Denaturation at Denaturation at
94degC94degC 1048708 1048708 Primer annealing Primer annealing
at Tm-5degCat Tm-5degC 1048708 1048708 Extension at 72degCExtension at 72degC
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
Templates for PCRTemplates for PCR
1048708 1048708 Dried bloodDried blood1048708 1048708 Semen stainsSemen stains1048708 1048708 Vaginal swabsVaginal swabs1048708 1048708 Single hairSingle hair1048708 1048708 Finger nail scrapingsFinger nail scrapings1048708 1048708 Egyptian mummiesEgyptian mummies1048708 1048708 Buccal SwabBuccal Swab1048708 1048708 Tooth brushesTooth brushes
Basic reactionBasic reaction
Thermocycling PCR machine1048707 Previously ndash need to overlay oil to prevent evaporation1048707 Automatically Change temperature1048707 Temperature gradient
The Basics of PCR CyclingThe Basics of PCR Cycling
bull bull 30ndash35 cycles each30ndash35 cycles each
comprisingcomprising
ndash ndash denaturation (95degC)denaturation (95degC)
30 sec30 sec
ndash ndash annealing (55ndash60degC)annealing (55ndash60degC)
30 sec30 sec
ndash ndash extension (72degC)extension (72degC)
time depends ontime depends on
product sizeproduct size
How many copiesHow many copies
bull bull No target products are made until the third No target products are made until the third cyclecycle
bull bull The accumulation is not strictly a doublingThe accumulation is not strictly a doubling
at each cycle in the early phaseat each cycle in the early phase
bull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 target
copies (~1times10copies (~1times1099))
How many cyclesHow many cycles
bull bull Increasing the cycleIncreasing the cyclenumber above ~35 hasnumber above ~35 haslittle positive effectlittle positive effectbull bull The plateau occurs whenThe plateau occurs whenndash ndash The reagents are depletedThe reagents are depletedndash ndash The products re-annealThe products re-annealndash ndash The polymerase isThe polymerase isdamageddamaged- Unwanted products- Unwanted productsaccumulateaccumulate
Basic reactionBasic reaction Oligonucleotide Oligonucleotide
primersprimers Design to flank the Design to flank the
desired sequencedesired sequence Steps include (30-Steps include (30-
40 steps)40 steps) 1048708 1048708 Denaturation at Denaturation at
94degC94degC 1048708 1048708 Primer annealing Primer annealing
at Tm-5degCat Tm-5degC 1048708 1048708 Extension at 72degCExtension at 72degC
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
Basic reactionBasic reaction
Thermocycling PCR machine1048707 Previously ndash need to overlay oil to prevent evaporation1048707 Automatically Change temperature1048707 Temperature gradient
The Basics of PCR CyclingThe Basics of PCR Cycling
bull bull 30ndash35 cycles each30ndash35 cycles each
comprisingcomprising
ndash ndash denaturation (95degC)denaturation (95degC)
30 sec30 sec
ndash ndash annealing (55ndash60degC)annealing (55ndash60degC)
30 sec30 sec
ndash ndash extension (72degC)extension (72degC)
time depends ontime depends on
product sizeproduct size
How many copiesHow many copies
bull bull No target products are made until the third No target products are made until the third cyclecycle
bull bull The accumulation is not strictly a doublingThe accumulation is not strictly a doubling
at each cycle in the early phaseat each cycle in the early phase
bull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 target
copies (~1times10copies (~1times1099))
How many cyclesHow many cycles
bull bull Increasing the cycleIncreasing the cyclenumber above ~35 hasnumber above ~35 haslittle positive effectlittle positive effectbull bull The plateau occurs whenThe plateau occurs whenndash ndash The reagents are depletedThe reagents are depletedndash ndash The products re-annealThe products re-annealndash ndash The polymerase isThe polymerase isdamageddamaged- Unwanted products- Unwanted productsaccumulateaccumulate
Basic reactionBasic reaction Oligonucleotide Oligonucleotide
primersprimers Design to flank the Design to flank the
desired sequencedesired sequence Steps include (30-Steps include (30-
40 steps)40 steps) 1048708 1048708 Denaturation at Denaturation at
94degC94degC 1048708 1048708 Primer annealing Primer annealing
at Tm-5degCat Tm-5degC 1048708 1048708 Extension at 72degCExtension at 72degC
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
The Basics of PCR CyclingThe Basics of PCR Cycling
bull bull 30ndash35 cycles each30ndash35 cycles each
comprisingcomprising
ndash ndash denaturation (95degC)denaturation (95degC)
30 sec30 sec
ndash ndash annealing (55ndash60degC)annealing (55ndash60degC)
30 sec30 sec
ndash ndash extension (72degC)extension (72degC)
time depends ontime depends on
product sizeproduct size
How many copiesHow many copies
bull bull No target products are made until the third No target products are made until the third cyclecycle
bull bull The accumulation is not strictly a doublingThe accumulation is not strictly a doubling
at each cycle in the early phaseat each cycle in the early phase
bull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 target
copies (~1times10copies (~1times1099))
How many cyclesHow many cycles
bull bull Increasing the cycleIncreasing the cyclenumber above ~35 hasnumber above ~35 haslittle positive effectlittle positive effectbull bull The plateau occurs whenThe plateau occurs whenndash ndash The reagents are depletedThe reagents are depletedndash ndash The products re-annealThe products re-annealndash ndash The polymerase isThe polymerase isdamageddamaged- Unwanted products- Unwanted productsaccumulateaccumulate
Basic reactionBasic reaction Oligonucleotide Oligonucleotide
primersprimers Design to flank the Design to flank the
desired sequencedesired sequence Steps include (30-Steps include (30-
40 steps)40 steps) 1048708 1048708 Denaturation at Denaturation at
94degC94degC 1048708 1048708 Primer annealing Primer annealing
at Tm-5degCat Tm-5degC 1048708 1048708 Extension at 72degCExtension at 72degC
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
How many copiesHow many copies
bull bull No target products are made until the third No target products are made until the third cyclecycle
bull bull The accumulation is not strictly a doublingThe accumulation is not strictly a doubling
at each cycle in the early phaseat each cycle in the early phase
bull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 target
copies (~1times10copies (~1times1099))
How many cyclesHow many cycles
bull bull Increasing the cycleIncreasing the cyclenumber above ~35 hasnumber above ~35 haslittle positive effectlittle positive effectbull bull The plateau occurs whenThe plateau occurs whenndash ndash The reagents are depletedThe reagents are depletedndash ndash The products re-annealThe products re-annealndash ndash The polymerase isThe polymerase isdamageddamaged- Unwanted products- Unwanted productsaccumulateaccumulate
Basic reactionBasic reaction Oligonucleotide Oligonucleotide
primersprimers Design to flank the Design to flank the
desired sequencedesired sequence Steps include (30-Steps include (30-
40 steps)40 steps) 1048708 1048708 Denaturation at Denaturation at
94degC94degC 1048708 1048708 Primer annealing Primer annealing
at Tm-5degCat Tm-5degC 1048708 1048708 Extension at 72degCExtension at 72degC
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
How many cyclesHow many cycles
bull bull Increasing the cycleIncreasing the cyclenumber above ~35 hasnumber above ~35 haslittle positive effectlittle positive effectbull bull The plateau occurs whenThe plateau occurs whenndash ndash The reagents are depletedThe reagents are depletedndash ndash The products re-annealThe products re-annealndash ndash The polymerase isThe polymerase isdamageddamaged- Unwanted products- Unwanted productsaccumulateaccumulate
Basic reactionBasic reaction Oligonucleotide Oligonucleotide
primersprimers Design to flank the Design to flank the
desired sequencedesired sequence Steps include (30-Steps include (30-
40 steps)40 steps) 1048708 1048708 Denaturation at Denaturation at
94degC94degC 1048708 1048708 Primer annealing Primer annealing
at Tm-5degCat Tm-5degC 1048708 1048708 Extension at 72degCExtension at 72degC
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
Basic reactionBasic reaction Oligonucleotide Oligonucleotide
primersprimers Design to flank the Design to flank the
desired sequencedesired sequence Steps include (30-Steps include (30-
40 steps)40 steps) 1048708 1048708 Denaturation at Denaturation at
94degC94degC 1048708 1048708 Primer annealing Primer annealing
at Tm-5degCat Tm-5degC 1048708 1048708 Extension at 72degCExtension at 72degC
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
Shortcut to pcranimatielnk
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
rtPCRrtPCR
Reverse Trascription PCR (RT-PCR)Reverse Trascription PCR (RT-PCR)
Use mRNA as a templateUse mRNA as a template Total cellular RNA - fasterTotal cellular RNA - faster Contamination of genomic Contamination of genomic
DNA ndash false resultDNA ndash false result Primer specific to exonsPrimer specific to exons Treat sample with DNaseTreat sample with DNase Can be quantitativeCan be quantitative
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
Multiplex PCRMultiplex PCR
Simultaneously modification of more thanSimultaneously modification of more than
one locus in the same reactionone locus in the same reaction Rapid and convenient ndash screeningRapid and convenient ndash screening Included different set of primersIncluded different set of primers
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
Quantitative or Real Time PCRQuantitative or Real Time PCR
Monitors the fluorescence emitted during theMonitors the fluorescence emitted during the
reaction as an indicator of amplicon productionreaction as an indicator of amplicon production during each PCR cycleduring each PCR cycle
1048708 1048708 The parameter CT (threshold cycle) is defined The parameter CT (threshold cycle) is defined asas
the cycle number at which the fluorescencethe cycle number at which the fluorescence
emission exceeds the fixed thresholdemission exceeds the fixed threshold
(background)(background)
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
Quantitative or Real Time PCRQuantitative or Real Time PCR
Three different fluorescence systemsHydrolysis probesHybridizing probesDNA binding agents SYBR-Green I
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
Molecular BeaconsMolecular Beacons
Uses FRETUses FRET
FFuorescence uorescence RResonance esonance EEnergy nergy TTransferransfer Uses two sequence specific Uses two sequence specific
oligonucleotides labeled with fluorescentoligonucleotides labeled with fluorescent
dyesdyes
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
In situ PCRIn situ PCR
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
Applications of PCRApplications of PCR
Mutation detection Mutation detection Diagnosis or screening of acquired Diagnosis or screening of acquired
diseases eg AIDS HBV amp HCVdiseases eg AIDS HBV amp HCV Prenatal diagnosisPrenatal diagnosis DNA profiling in forensic science DNA profiling in forensic science Quantitation of mRNA in cells or Quantitation of mRNA in cells or
tissues tissues
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
Problems with PCRProblems with PCR
ContaminationContamination Theoretically one molecule can amplifyTheoretically one molecule can amplify Takes one mismatch early on to amplifyTakes one mismatch early on to amplify
the wrong fragmentthe wrong fragment
Dr Naglaa FathyDr Naglaa Fathy
Dr Naglaa FathyDr Naglaa Fathy