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Polymerase Chain Reaction
Invented by Kary Mullis
1048708 Mullis and Faloona 1987 Specificsynthesis of DNA in vitro via apolymerase-catalyzed chain reaction
1048708 Nobel Prize 1993
ldquoI was working for Cetus making oligonucleotidesThey were heady times Biotechnology was in flowerand one spring night while the California buckeyeswere also in flower I came across the polymerasechain reaction I was driving with Jennifer Barnett toa cabin I had been building in northern CaliforniaShe and I had worked and lived together for twoyears She was an inspiration to me during that timeas only a woman with brains in the bloom of herwomanhood can be That morning she had no ideawhat had just happened I had an inkling It was thefirst day of the rest of my liferdquo- from Karry Mullisrsquos autobiography at the Nobel e-Museum
Specifically targets and amplifies aSINGLE sequence from within a complexmixture of DNAHow is this different from cloning
Takes advantage of basicrequirements of replication1048708 A DNA template1048708 Nucleotides1048708 Primers1048708 polymerasePCR is DNA replication in a test tube
Primers1048708 Must have some information aboutsequence flanking your target1048708 Primers provide specificity
1048708 Complementary to opposite strands with 3rsquoends pointing towards each other1048708 Should have similar melting temperatures1048708 Be in vast excess
Melting temperature
1048708 TmoC = 2(AT) + 4(GC)1048708 TmoC Temperature at whichhalf possible H bonds areformed
Steps of PCR
Annealing then primer elongation
Thermocycling
1048708 94 degrees
1048708 55 degrees
1048708 70 degree
Heat-stable polymerase is vitalto the ease of the processhellip
Problems with Taq
Automated long time agohellip
Problems with PCR
1048708 Contamination
1048708 Takes one mismatch early on to amplify the wrong fragment
Invented by Kary Mullis
1048708 Mullis and Faloona 1987 Specificsynthesis of DNA in vitro via apolymerase-catalyzed chain reaction
1048708 Nobel Prize 1993
ldquoI was working for Cetus making oligonucleotidesThey were heady times Biotechnology was in flowerand one spring night while the California buckeyeswere also in flower I came across the polymerasechain reaction I was driving with Jennifer Barnett toa cabin I had been building in northern CaliforniaShe and I had worked and lived together for twoyears She was an inspiration to me during that timeas only a woman with brains in the bloom of herwomanhood can be That morning she had no ideawhat had just happened I had an inkling It was thefirst day of the rest of my liferdquo- from Karry Mullisrsquos autobiography at the Nobel e-Museum
Specifically targets and amplifies aSINGLE sequence from within a complexmixture of DNAHow is this different from cloning
Takes advantage of basicrequirements of replication1048708 A DNA template1048708 Nucleotides1048708 Primers1048708 polymerasePCR is DNA replication in a test tube
Primers1048708 Must have some information aboutsequence flanking your target1048708 Primers provide specificity
1048708 Complementary to opposite strands with 3rsquoends pointing towards each other1048708 Should have similar melting temperatures1048708 Be in vast excess
Melting temperature
1048708 TmoC = 2(AT) + 4(GC)1048708 TmoC Temperature at whichhalf possible H bonds areformed
Steps of PCR
Annealing then primer elongation
Thermocycling
1048708 94 degrees
1048708 55 degrees
1048708 70 degree
Heat-stable polymerase is vitalto the ease of the processhellip
Problems with Taq
Automated long time agohellip
Problems with PCR
1048708 Contamination
1048708 Takes one mismatch early on to amplify the wrong fragment
ldquoI was working for Cetus making oligonucleotidesThey were heady times Biotechnology was in flowerand one spring night while the California buckeyeswere also in flower I came across the polymerasechain reaction I was driving with Jennifer Barnett toa cabin I had been building in northern CaliforniaShe and I had worked and lived together for twoyears She was an inspiration to me during that timeas only a woman with brains in the bloom of herwomanhood can be That morning she had no ideawhat had just happened I had an inkling It was thefirst day of the rest of my liferdquo- from Karry Mullisrsquos autobiography at the Nobel e-Museum
Specifically targets and amplifies aSINGLE sequence from within a complexmixture of DNAHow is this different from cloning
Takes advantage of basicrequirements of replication1048708 A DNA template1048708 Nucleotides1048708 Primers1048708 polymerasePCR is DNA replication in a test tube
Primers1048708 Must have some information aboutsequence flanking your target1048708 Primers provide specificity
1048708 Complementary to opposite strands with 3rsquoends pointing towards each other1048708 Should have similar melting temperatures1048708 Be in vast excess
Melting temperature
1048708 TmoC = 2(AT) + 4(GC)1048708 TmoC Temperature at whichhalf possible H bonds areformed
Steps of PCR
Annealing then primer elongation
Thermocycling
1048708 94 degrees
1048708 55 degrees
1048708 70 degree
Heat-stable polymerase is vitalto the ease of the processhellip
Problems with Taq
Automated long time agohellip
Problems with PCR
1048708 Contamination
1048708 Takes one mismatch early on to amplify the wrong fragment
Specifically targets and amplifies aSINGLE sequence from within a complexmixture of DNAHow is this different from cloning
Takes advantage of basicrequirements of replication1048708 A DNA template1048708 Nucleotides1048708 Primers1048708 polymerasePCR is DNA replication in a test tube
Primers1048708 Must have some information aboutsequence flanking your target1048708 Primers provide specificity
1048708 Complementary to opposite strands with 3rsquoends pointing towards each other1048708 Should have similar melting temperatures1048708 Be in vast excess
Melting temperature
1048708 TmoC = 2(AT) + 4(GC)1048708 TmoC Temperature at whichhalf possible H bonds areformed
Steps of PCR
Annealing then primer elongation
Thermocycling
1048708 94 degrees
1048708 55 degrees
1048708 70 degree
Heat-stable polymerase is vitalto the ease of the processhellip
Problems with Taq
Automated long time agohellip
Problems with PCR
1048708 Contamination
1048708 Takes one mismatch early on to amplify the wrong fragment
Takes advantage of basicrequirements of replication1048708 A DNA template1048708 Nucleotides1048708 Primers1048708 polymerasePCR is DNA replication in a test tube
Primers1048708 Must have some information aboutsequence flanking your target1048708 Primers provide specificity
1048708 Complementary to opposite strands with 3rsquoends pointing towards each other1048708 Should have similar melting temperatures1048708 Be in vast excess
Melting temperature
1048708 TmoC = 2(AT) + 4(GC)1048708 TmoC Temperature at whichhalf possible H bonds areformed
Steps of PCR
Annealing then primer elongation
Thermocycling
1048708 94 degrees
1048708 55 degrees
1048708 70 degree
Heat-stable polymerase is vitalto the ease of the processhellip
Problems with Taq
Automated long time agohellip
Problems with PCR
1048708 Contamination
1048708 Takes one mismatch early on to amplify the wrong fragment
Primers1048708 Must have some information aboutsequence flanking your target1048708 Primers provide specificity
1048708 Complementary to opposite strands with 3rsquoends pointing towards each other1048708 Should have similar melting temperatures1048708 Be in vast excess
Melting temperature
1048708 TmoC = 2(AT) + 4(GC)1048708 TmoC Temperature at whichhalf possible H bonds areformed
Steps of PCR
Annealing then primer elongation
Thermocycling
1048708 94 degrees
1048708 55 degrees
1048708 70 degree
Heat-stable polymerase is vitalto the ease of the processhellip
Problems with Taq
Automated long time agohellip
Problems with PCR
1048708 Contamination
1048708 Takes one mismatch early on to amplify the wrong fragment
1048708 Complementary to opposite strands with 3rsquoends pointing towards each other1048708 Should have similar melting temperatures1048708 Be in vast excess
Melting temperature
1048708 TmoC = 2(AT) + 4(GC)1048708 TmoC Temperature at whichhalf possible H bonds areformed
Steps of PCR
Annealing then primer elongation
Thermocycling
1048708 94 degrees
1048708 55 degrees
1048708 70 degree
Heat-stable polymerase is vitalto the ease of the processhellip
Problems with Taq
Automated long time agohellip
Problems with PCR
1048708 Contamination
1048708 Takes one mismatch early on to amplify the wrong fragment
Melting temperature
1048708 TmoC = 2(AT) + 4(GC)1048708 TmoC Temperature at whichhalf possible H bonds areformed
Steps of PCR
Annealing then primer elongation
Thermocycling
1048708 94 degrees
1048708 55 degrees
1048708 70 degree
Heat-stable polymerase is vitalto the ease of the processhellip
Problems with Taq
Automated long time agohellip
Problems with PCR
1048708 Contamination
1048708 Takes one mismatch early on to amplify the wrong fragment
Steps of PCR
Annealing then primer elongation
Thermocycling
1048708 94 degrees
1048708 55 degrees
1048708 70 degree
Heat-stable polymerase is vitalto the ease of the processhellip
Problems with Taq
Automated long time agohellip
Problems with PCR
1048708 Contamination
1048708 Takes one mismatch early on to amplify the wrong fragment
Annealing then primer elongation
Thermocycling
1048708 94 degrees
1048708 55 degrees
1048708 70 degree
Heat-stable polymerase is vitalto the ease of the processhellip
Problems with Taq
Automated long time agohellip
Problems with PCR
1048708 Contamination
1048708 Takes one mismatch early on to amplify the wrong fragment
Thermocycling
1048708 94 degrees
1048708 55 degrees
1048708 70 degree
Heat-stable polymerase is vitalto the ease of the processhellip
Problems with Taq
Automated long time agohellip
Problems with PCR
1048708 Contamination
1048708 Takes one mismatch early on to amplify the wrong fragment
Heat-stable polymerase is vitalto the ease of the processhellip
Problems with Taq
Automated long time agohellip
Problems with PCR
1048708 Contamination
1048708 Takes one mismatch early on to amplify the wrong fragment
Problems with Taq
Automated long time agohellip
Problems with PCR
1048708 Contamination
1048708 Takes one mismatch early on to amplify the wrong fragment
Automated long time agohellip
Problems with PCR
1048708 Contamination
1048708 Takes one mismatch early on to amplify the wrong fragment
Problems with PCR
1048708 Contamination
1048708 Takes one mismatch early on to amplify the wrong fragment
