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454 sequencing

Pirossequenciamento
by

Bruno Karolski

on 30 August 2011

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Transcript of 454 sequencing

454 Sequencing
Tudo começa....
Preparação da biblioteca
Preparando as beads de DNA
Pré sequenciamento
Captura da biblioteca
Ligação streptavidina - biotina
Titulação da biblioteca
600.000 beads
0,5 2 4 8 CPB (cópias por bead)
PCR de emulsão
"1 bead 1 read"
Amplificação da mesma molécula de DNA
Cada gota de óleo - 1 minireator
50 a 100 micrometros
Enriquecimento
Três tipos de beads
Pool de beads após a emPCR
Ligação biotina-estreptavidina em beads magnéticas
Separação magnética das beads com DNA
$
Sequenciamento
Primer annealing
Cromatograma
Solid Sequencing
Quebra do DNA e ligação
1
2
PCR de emulsão
3
Imobilização das beads em placas de vidro
4
Incorporação das sequências marcadas
5
Montagem das sequências
Leitura dupla de cada base
Ilumina Sequencing
Sequenciamento de segunda geração
Fim
Full transcript