polymerase chain reaction - bmg.fc.ul.ptbmg.fc.ul.pt/disciplinas/gbm/aulas/5pcr.pdf · denature dna...
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![Page 1: Polymerase Chain Reaction - bmg.fc.ul.ptbmg.fc.ul.pt/Disciplinas/GBM/aulas/5PCR.pdf · Denature DNA sample to separate DNA strands (94ºC, 5 min) Primer bind to DNA strands (30-65ºC,](https://reader033.vdocuments.net/reader033/viewer/2022060213/5f0543107e708231d4121682/html5/thumbnails/1.jpg)
PCRPolymerase Chain Reaction
Selective amplification
of a
SPECIFIC target DNA sequence,
within a heterogenous collection of DNA sequences
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![Page 3: Polymerase Chain Reaction - bmg.fc.ul.ptbmg.fc.ul.pt/Disciplinas/GBM/aulas/5PCR.pdf · Denature DNA sample to separate DNA strands (94ºC, 5 min) Primer bind to DNA strands (30-65ºC,](https://reader033.vdocuments.net/reader033/viewer/2022060213/5f0543107e708231d4121682/html5/thumbnails/3.jpg)
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Each new strand now begins withone primer sequence and endswith the primer-binding sequencefor the other primer
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Exponential copies of template DNA
2n
n- nº of cycles
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Cooper 3.27
Denaturation(Strand separation)
Extension(DNA synthesis)
Annealing(Primer hybridization)
Stepsin each cycle
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Denature DNA sampleto separate DNA strands(94ºC, 5 min)
Primer bind toDNA strands(30-65ºC, 30 s)
Primers bind toDNA strands(30-65ºC, 30 s)
PCR conditions
ANNEALING
DENATURATION
EXTENSION
Denaturate to separateDNA strands(94ºC, 30 s)
Denaturate to separateDNA strands(94ºC, 30 s)
DENATURATION
Polymerase synthesizesnew DNA strands(65º-75ºC, 2-5 min)
Polymerase synthesizesnew DNA strands(65º-75ºC, 2-5 min)
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Perfil térmico de um PCR
1-2 min30 s-1 min
DESNATURAÇÃO EMPARELHAMENTO(Annealing)
EXTENSÃO
30 s-5 min*2
*2- mto variável; depende do nº de pb do fragmento de DNA a amplificar e da processividade da enzima
*1
*1- depende do Tm (temperatura de fusão) dos primers
30 s-1 min
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CONDIÇÕES de REACÇÃO de uma experiência de PCR
DNAPrimer 1Primer 2dNTPsDNA polymeraseBuffer + Mg 2+
H20
PCR reaction
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Algumas DNA polimerases usadas em PCR
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Características da Taq DNA polimerase
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Commercial DNA polymerasesused in PCR
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Choosing sequence primers
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PCR applicationsex.
• Amplification of specific DNA fragments to clone, or to isolate a specific clone
• Detection of polymorphisms
• Distinction between alleles
• Phylogenetic studies
• …
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VNTR detection by PCRI
Ex: D1S80
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VNTR detection by PCRII
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VNTR detection by PCR using multiple primersMultiplex reaction
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PCR used to isolate a specific clone
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Amplification of 16S rRNA gene in order to performphylogenetic studies