PCR Polymerase Chain Reaction Selective amplification of a SPECIFIC target DNA sequence, within a heterogenous collection of DNA sequences
PCRPolymerase Chain Reaction
Selective amplification
of a
SPECIFIC target DNA sequence,
within a heterogenous collection of DNA sequences
Each new strand now begins withone primer sequence and endswith the primer-binding sequencefor the other primer
Exponential copies of template DNA
2n
n- nº of cycles
Cooper 3.27
Denaturation(Strand separation)
Extension(DNA synthesis)
Annealing(Primer hybridization)
Stepsin each cycle
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)
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
CONDIÇÕES de REACÇÃO de uma experiência de PCR
DNAPrimer 1Primer 2dNTPsDNA polymeraseBuffer + Mg 2+
H20
PCR reaction
Algumas DNA polimerases usadas em PCR
Características da Taq DNA polimerase
Commercial DNA polymerasesused in PCR
Choosing sequence primers
PCR applicationsex.
• Amplification of specific DNA fragments to clone, or to isolate a specific clone
• Detection of polymorphisms
• Distinction between alleles
• Phylogenetic studies
• …
VNTR detection by PCRI
Ex: D1S80
VNTR detection by PCRII
VNTR detection by PCR using multiple primersMultiplex reaction
PCR used to isolate a specific clone
Amplification of 16S rRNA gene in order to performphylogenetic studies