polymerase chain reaction - bmg.fc.ul.ptbmg.fc.ul.pt/disciplinas/gbm/aulas/5pcr.pdf · denature dna...

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