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بسم الله الرحمن الرحيم

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بسم الله الرحمن الرحيم. The Polymerase Chain Reaction (PCR). By Dr. NAGLAA FATHY Lecturer of Biochemistry & Molecular Biology Faculty of Medicine Benha University E-mail : [email protected] [email protected]. What is the Polymerase Chain Reaction?. - PowerPoint PPT Presentation

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Page 1: بسم الله الرحمن الرحيم

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 reactionNobel 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 aSINGLE 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

PrimersPrimersMust 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 Annealing3 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 permanentlydestroyed at 94ordmCdestroyed at 94ordmC OptimalOptimaltemperature 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 reactionThermocycling 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 eachcomprisingcomprisingndash ndash denaturation (95degC)denaturation (95degC)30 sec30 secndash ndash annealing (55ndash60degC)annealing (55ndash60degC)30 sec30 secndash ndash extension (72degC)extension (72degC)time depends ontime depends onproduct 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 doublingat each cycle in the early phaseat each cycle in the early phasebull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 targetcopies (~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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 2: بسم الله الرحمن الرحيم

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 reactionNobel 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 aSINGLE 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

PrimersPrimersMust 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 Annealing3 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 permanentlydestroyed at 94ordmCdestroyed at 94ordmC OptimalOptimaltemperature 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 reactionThermocycling 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 eachcomprisingcomprisingndash ndash denaturation (95degC)denaturation (95degC)30 sec30 secndash ndash annealing (55ndash60degC)annealing (55ndash60degC)30 sec30 secndash ndash extension (72degC)extension (72degC)time depends ontime depends onproduct 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 doublingat each cycle in the early phaseat each cycle in the early phasebull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 targetcopies (~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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 3: بسم الله الرحمن الرحيم

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 reactionNobel 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 aSINGLE 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

PrimersPrimersMust 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 Annealing3 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 permanentlydestroyed at 94ordmCdestroyed at 94ordmC OptimalOptimaltemperature 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 reactionThermocycling 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 eachcomprisingcomprisingndash ndash denaturation (95degC)denaturation (95degC)30 sec30 secndash ndash annealing (55ndash60degC)annealing (55ndash60degC)30 sec30 secndash ndash extension (72degC)extension (72degC)time depends ontime depends onproduct 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 doublingat each cycle in the early phaseat each cycle in the early phasebull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 targetcopies (~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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 4: بسم الله الرحمن الرحيم

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 aSINGLE 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

PrimersPrimersMust 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 Annealing3 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 permanentlydestroyed at 94ordmCdestroyed at 94ordmC OptimalOptimaltemperature 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 reactionThermocycling 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 eachcomprisingcomprisingndash ndash denaturation (95degC)denaturation (95degC)30 sec30 secndash ndash annealing (55ndash60degC)annealing (55ndash60degC)30 sec30 secndash ndash extension (72degC)extension (72degC)time depends ontime depends onproduct 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 doublingat each cycle in the early phaseat each cycle in the early phasebull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 targetcopies (~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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 5: بسم الله الرحمن الرحيم

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 aSINGLE 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

PrimersPrimersMust 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 Annealing3 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 permanentlydestroyed at 94ordmCdestroyed at 94ordmC OptimalOptimaltemperature 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 reactionThermocycling 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 eachcomprisingcomprisingndash ndash denaturation (95degC)denaturation (95degC)30 sec30 secndash ndash annealing (55ndash60degC)annealing (55ndash60degC)30 sec30 secndash ndash extension (72degC)extension (72degC)time depends ontime depends onproduct 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 doublingat each cycle in the early phaseat each cycle in the early phasebull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 targetcopies (~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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 6: بسم الله الرحمن الرحيم

PCRPCR

Specifically targets and amplifies aSpecifically targets and amplifies aSINGLE 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

PrimersPrimersMust 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 Annealing3 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 permanentlydestroyed at 94ordmCdestroyed at 94ordmC OptimalOptimaltemperature 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 reactionThermocycling 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 eachcomprisingcomprisingndash ndash denaturation (95degC)denaturation (95degC)30 sec30 secndash ndash annealing (55ndash60degC)annealing (55ndash60degC)30 sec30 secndash ndash extension (72degC)extension (72degC)time depends ontime depends onproduct 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 doublingat each cycle in the early phaseat each cycle in the early phasebull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 targetcopies (~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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 7: بسم الله الرحمن الرحيم

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

PrimersPrimersMust 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 Annealing3 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 permanentlydestroyed at 94ordmCdestroyed at 94ordmC OptimalOptimaltemperature 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 reactionThermocycling 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 eachcomprisingcomprisingndash ndash denaturation (95degC)denaturation (95degC)30 sec30 secndash ndash annealing (55ndash60degC)annealing (55ndash60degC)30 sec30 secndash ndash extension (72degC)extension (72degC)time depends ontime depends onproduct 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 doublingat each cycle in the early phaseat each cycle in the early phasebull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 targetcopies (~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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 8: بسم الله الرحمن الرحيم

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

PrimersPrimersMust 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 Annealing3 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 permanentlydestroyed at 94ordmCdestroyed at 94ordmC OptimalOptimaltemperature 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 reactionThermocycling 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 eachcomprisingcomprisingndash ndash denaturation (95degC)denaturation (95degC)30 sec30 secndash ndash annealing (55ndash60degC)annealing (55ndash60degC)30 sec30 secndash ndash extension (72degC)extension (72degC)time depends ontime depends onproduct 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 doublingat each cycle in the early phaseat each cycle in the early phasebull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 targetcopies (~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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 9: بسم الله الرحمن الرحيم

PRIMERSPRIMERS

Primers short ssDNA sequencescomplementary to border of sequence of interest

PrimersPrimersMust 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 Annealing3 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 permanentlydestroyed at 94ordmCdestroyed at 94ordmC OptimalOptimaltemperature 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 reactionThermocycling 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 eachcomprisingcomprisingndash ndash denaturation (95degC)denaturation (95degC)30 sec30 secndash ndash annealing (55ndash60degC)annealing (55ndash60degC)30 sec30 secndash ndash extension (72degC)extension (72degC)time depends ontime depends onproduct 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 doublingat each cycle in the early phaseat each cycle in the early phasebull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 targetcopies (~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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 10: بسم الله الرحمن الرحيم

PrimersPrimersMust 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 Annealing3 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 permanentlydestroyed at 94ordmCdestroyed at 94ordmC OptimalOptimaltemperature 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 reactionThermocycling 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 eachcomprisingcomprisingndash ndash denaturation (95degC)denaturation (95degC)30 sec30 secndash ndash annealing (55ndash60degC)annealing (55ndash60degC)30 sec30 secndash ndash extension (72degC)extension (72degC)time depends ontime depends onproduct 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 doublingat each cycle in the early phaseat each cycle in the early phasebull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 targetcopies (~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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 11: بسم الله الرحمن الرحيم

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 Annealing3 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 permanentlydestroyed at 94ordmCdestroyed at 94ordmC OptimalOptimaltemperature 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 reactionThermocycling 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 eachcomprisingcomprisingndash ndash denaturation (95degC)denaturation (95degC)30 sec30 secndash ndash annealing (55ndash60degC)annealing (55ndash60degC)30 sec30 secndash ndash extension (72degC)extension (72degC)time depends ontime depends onproduct 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 doublingat each cycle in the early phaseat each cycle in the early phasebull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 targetcopies (~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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 12: بسم الله الرحمن الرحيم

PCRPCRRegion of interestbetween primers

2 Anneal

3 Extend

Taq polymerase enzymatic extension

PCRRepeated Cycles ofRepeated Cycles of

1 Denaturation 1 Denaturation 2 Annealing2 Annealing3 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 permanentlydestroyed at 94ordmCdestroyed at 94ordmC OptimalOptimaltemperature 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 reactionThermocycling 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 eachcomprisingcomprisingndash ndash denaturation (95degC)denaturation (95degC)30 sec30 secndash ndash annealing (55ndash60degC)annealing (55ndash60degC)30 sec30 secndash ndash extension (72degC)extension (72degC)time depends ontime depends onproduct 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 doublingat each cycle in the early phaseat each cycle in the early phasebull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 targetcopies (~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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 13: بسم الله الرحمن الرحيم

PCRRepeated Cycles ofRepeated Cycles of

1 Denaturation 1 Denaturation 2 Annealing2 Annealing3 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 permanentlydestroyed at 94ordmCdestroyed at 94ordmC OptimalOptimaltemperature 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 reactionThermocycling 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 eachcomprisingcomprisingndash ndash denaturation (95degC)denaturation (95degC)30 sec30 secndash ndash annealing (55ndash60degC)annealing (55ndash60degC)30 sec30 secndash ndash extension (72degC)extension (72degC)time depends ontime depends onproduct 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 doublingat each cycle in the early phaseat each cycle in the early phasebull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 targetcopies (~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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 14: بسم الله الرحمن الرحيم

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 permanentlydestroyed at 94ordmCdestroyed at 94ordmC OptimalOptimaltemperature 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 reactionThermocycling 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 eachcomprisingcomprisingndash ndash denaturation (95degC)denaturation (95degC)30 sec30 secndash ndash annealing (55ndash60degC)annealing (55ndash60degC)30 sec30 secndash ndash extension (72degC)extension (72degC)time depends ontime depends onproduct 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 doublingat each cycle in the early phaseat each cycle in the early phasebull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 targetcopies (~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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 15: بسم الله الرحمن الرحيم

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 permanentlydestroyed at 94ordmCdestroyed at 94ordmC OptimalOptimaltemperature 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 reactionThermocycling 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 eachcomprisingcomprisingndash ndash denaturation (95degC)denaturation (95degC)30 sec30 secndash ndash annealing (55ndash60degC)annealing (55ndash60degC)30 sec30 secndash ndash extension (72degC)extension (72degC)time depends ontime depends onproduct 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 doublingat each cycle in the early phaseat each cycle in the early phasebull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 targetcopies (~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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 16: بسم الله الرحمن الرحيم

Thermus aquaticusThermus aquaticus

Thermus aquaticus from hot springs inYellowstone National Park USA

The ThermusThe Thermusaquaticus DNAaquaticus DNA

polymerasepolymerase

TaqTaq Not permanentlyNot permanentlydestroyed at 94ordmCdestroyed at 94ordmC OptimalOptimaltemperature 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 reactionThermocycling 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 eachcomprisingcomprisingndash ndash denaturation (95degC)denaturation (95degC)30 sec30 secndash ndash annealing (55ndash60degC)annealing (55ndash60degC)30 sec30 secndash ndash extension (72degC)extension (72degC)time depends ontime depends onproduct 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 doublingat each cycle in the early phaseat each cycle in the early phasebull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 targetcopies (~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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 17: بسم الله الرحمن الرحيم

The ThermusThe Thermusaquaticus DNAaquaticus DNA

polymerasepolymerase

TaqTaq Not permanentlyNot permanentlydestroyed at 94ordmCdestroyed at 94ordmC OptimalOptimaltemperature 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 reactionThermocycling 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 eachcomprisingcomprisingndash ndash denaturation (95degC)denaturation (95degC)30 sec30 secndash ndash annealing (55ndash60degC)annealing (55ndash60degC)30 sec30 secndash ndash extension (72degC)extension (72degC)time depends ontime depends onproduct 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 doublingat each cycle in the early phaseat each cycle in the early phasebull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 targetcopies (~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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 18: بسم الله الرحمن الرحيم

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 reactionThermocycling 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 eachcomprisingcomprisingndash ndash denaturation (95degC)denaturation (95degC)30 sec30 secndash ndash annealing (55ndash60degC)annealing (55ndash60degC)30 sec30 secndash ndash extension (72degC)extension (72degC)time depends ontime depends onproduct 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 doublingat each cycle in the early phaseat each cycle in the early phasebull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 targetcopies (~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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 19: بسم الله الرحمن الرحيم

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 reactionThermocycling 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 eachcomprisingcomprisingndash ndash denaturation (95degC)denaturation (95degC)30 sec30 secndash ndash annealing (55ndash60degC)annealing (55ndash60degC)30 sec30 secndash ndash extension (72degC)extension (72degC)time depends ontime depends onproduct 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 doublingat each cycle in the early phaseat each cycle in the early phasebull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 targetcopies (~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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 20: بسم الله الرحمن الرحيم

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 reactionThermocycling 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 eachcomprisingcomprisingndash ndash denaturation (95degC)denaturation (95degC)30 sec30 secndash ndash annealing (55ndash60degC)annealing (55ndash60degC)30 sec30 secndash ndash extension (72degC)extension (72degC)time depends ontime depends onproduct 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 doublingat each cycle in the early phaseat each cycle in the early phasebull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 targetcopies (~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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 21: بسم الله الرحمن الرحيم

Basic reactionBasic reactionThermocycling 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 eachcomprisingcomprisingndash ndash denaturation (95degC)denaturation (95degC)30 sec30 secndash ndash annealing (55ndash60degC)annealing (55ndash60degC)30 sec30 secndash ndash extension (72degC)extension (72degC)time depends ontime depends onproduct 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 doublingat each cycle in the early phaseat each cycle in the early phasebull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 targetcopies (~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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 22: بسم الله الرحمن الرحيم

The Basics of PCR CyclingThe Basics of PCR Cycling

bull bull 30ndash35 cycles each30ndash35 cycles eachcomprisingcomprisingndash ndash denaturation (95degC)denaturation (95degC)30 sec30 secndash ndash annealing (55ndash60degC)annealing (55ndash60degC)30 sec30 secndash ndash extension (72degC)extension (72degC)time depends ontime depends onproduct 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 doublingat each cycle in the early phaseat each cycle in the early phasebull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 targetcopies (~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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 23: بسم الله الرحمن الرحيم

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 doublingat each cycle in the early phaseat each cycle in the early phasebull bull At 30 cycles there are 1073741764 targetAt 30 cycles there are 1073741764 targetcopies (~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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 24: بسم الله الرحمن الرحيم

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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 25: بسم الله الرحمن الرحيم

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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 26: بسم الله الرحمن الرحيم

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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 27: بسم الله الرحمن الرحيم

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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 28: بسم الله الرحمن الرحيم

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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 29: بسم الله الرحمن الرحيم

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 cycle1048708 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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 30: بسم الله الرحمن الرحيم

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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 31: بسم الله الرحمن الرحيم

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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 32: بسم الله الرحمن الرحيم

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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 33: بسم الله الرحمن الرحيم

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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 34: بسم الله الرحمن الرحيم

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 Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37
Page 35: بسم الله الرحمن الرحيم

Dr Naglaa FathyDr Naglaa Fathy

  • The Polymerase Chain Reaction (PCR)
  • What is the Polymerase Chain Reaction
  • Invented by Kary Mullis
  • Kary Mullis
  • Did He Really Invent PCR
  • PCR
  • Amplify DNA PCR
  • Takes advantage of basic requirements of replication
  • PRIMERS
  • Primers
  • Slide 12
  • PCR
  • Slide 14
  • Melting temperature
  • Heat-stable polymerase is vital to the ease of the processhellip
  • The Thermus aquaticus DNA polymerase
  • Problems with Taq
  • Termplates for PCR
  • Templates for PCR
  • Basic reaction
  • The Basics of PCR Cycling
  • How many copies
  • How many cycles
  • Slide 26
  • Slide 27
  • rtPCR
  • Multiplex PCR
  • Slide 30
  • Quantitative or Real Time PCR
  • Quantitative or Real Time PCR
  • Molecular Beacons
  • In situ PCR
  • Applications of PCR
  • Problems with PCR
  • Slide 37