Download - Sequenciamento
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Seqüenciamento
Técnica pela qual a ordem exata dos nucleotídeos em um segmento de
DNA pode ser determinada.
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Método de Sanger-Coulson: nucleotídeos terminadores de cadeia
• Necessita de DNA fita simples
• Utiliza apenas um dos primers
• Utiliza dNTPs marcados radioativamente (32P ou 35S-dNTP)
• Utiliza didesoxinucleotídeos (ddNTPs)
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Seqüenciamento automático de DNA
• Utilização de marcadores fluorescentes ligados a cada um dos ddNTPs
• Ocorre apenas uma reação de seqüenciamento como os 4 ddNTPs
• Moléculas terminadas com ddNTPs diferentes podem ser identificadas por meio de seus sinais fluorescentes distintos
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Seqüenciamento automático de DNA
• A amostra pós-PCR é aplicada num gel ou em um capilar de poliacrilamida e então passado por um detector de fluorescência
• O detector identifica o sinal fluorescente emitido pelas bandas e transmite os dados para o computador
• O computador converte a informação na seqüência de DNA apropriada.
• A seqüência pode ser impressa para análise ou enviada para um arquivo para ser analisada por softwares específicos
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Seqüenciamento automático de DNA
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Análise do seqüenciamento
TAAAGTCCAGAGCCACCATCCTGGCCGCAGGTAGCCGCTGGTCTCCGGGGTCATCTAGCGTCCGACCTCGGAGCGTGCATCCCAGGACGGTGACACCCTCCCCTGAGCTTCGGCGGCCACACAACCTTCCAGGGAGCTACAATGGAAGAATCACAGGCAGAACTCAATGTGGAGCCCCCTCTGAGTCAGGAGACATTTTCCGACTTGTGGAACCTACTTCCTGAAAATAACCTTCTGTCCTCCGAGCTCTCCGCACCCGTGGATGACCTGCTCCCGTACACAGATGTTGCCACCTGGCTGGATGAATGTCCGAATGAAGCGCCCCAAATGCCAGAGCCTTCTGCCCCAGCTGCCCCGCCACCAGCCACCCCAGCACCAGCCACCTCCTGGCCCCTGTCGTCCTTTGTCCCCTCCCAGAAGACCTACCCTGGCAATTACGGTTTCCGTCTAGGGTTCCTGCAATCCGGAACAGCCAAGTCCGTGACCTGCACGTATTCCCCTTCCCTTAACAAGCTGTTCTGCCAGCTGGCCAAGACCTGCCCAGTGCAGCTGTGGGTCGACTCGCCACCCCCGCCCGGCACCCGCGTCCGCGCCATGGCCATCTACAAGAAGTTGGAGCACATGACGGAGGTTGTGAGGCGTTGTCCCCACCATGAGCGCTCCTCTGACTATAGCGATGGTCTGGCCCCTCCTCAGCACCTTATCCGGGTGGAAGGGAATTTACGCGCGGAGTATTTGGACGACCGGAACACCTTTAGACACAGTGTGGTGGTGCCCTATGAGTCCCCCGAGATCGACTCTGAGTGCACCACCATCCACTACAATTTCATGTGTAACAGCTCCTGCATGGGGGGCATGAACCGGCGGCCCATCCTCACCATCATCACACTGGAAGACTCTTGTGGTAACCTGCTGGGACGGAACAGCTTTGAGGTGCGTGTTTGTGCCTGTCCTGGGAGAGACCGCCGCACTGAGGAAGAAAATCTTCGCAAGAAGGGGCAGTCTTGCCCCGAGCCACCCCCTAGGAGCACTAAGCGAGCACTGCCTACCAACACCAGCTCCTCTCCACAGCCAAAGAAGAAACCACTGGATGGAGAGTACTTCACTCTTCAGATCCGTGGGTTTAAACGCTATGAGATGTTCCGAGAGCTGAATGATGCCTTGGAGCTGAAGGATGCTCTGGATGGAAGGGAACCAGGGGAAAGCAGGGCTCACTCTAGCCACCTGAAGTCTAAGAAGAGGCCTTCTCCCTCCTGCCATAAAAAACCAATGCTCAAGAGAGAGGGACCTGACTCAGACTGATGTCTTCTGCTTCCTGTCACCTGTTGACATCCCCACCCATATCCCCTGCTCCTTCCCTGGCATTTTTTAAGACTCAGGTGCTTAGACCTCTGCTTGGTGCAGGTGCGCCTTAGAAACCCCCTGGGAGTCTTCATCTGCTCAGCCTGTGGCTGTGCTAAAGAGGCAGACCTGCGCTCGTGGTTTCTGGGGATTTGGGGGGTGCTGGCGCTTTCCAGCTTAGCTTTCAAGGTTTTTACTGTGAGCCGTGTTCGGGAGAGGTCAGAATGTGTTCCTGCATGTGAGGAACATCTTATAACCAGCCATACACTGGGTTGGAAAACTCAGTCCTCTGCCATACTAGCCAGGGAAGCTATCTTAAGTTGTTCGATTTTCTCCTGACTTCATTGTTCACATCTATAAAAAACTGATAATCATACCTACCTCACGGGGGGTTTGTTGTGAGGGTTAATGAAATAATGTATGTCTGGCCCCAAAACCATTATTTGATTACATAATGTCTGGGACTTAGTGCCTTTTGTGGGTGCTGGTTCTTTGTCCCCCTGGGTGGATGATTTGACAGTTTCTGACTGAATGGCTGGTTAAAGGGAAGAAGTCTCCAAGTCCCTTCTCTCCTGGCCCTGCCAGATCATAACCCCTTGGTGAACCTCAGTGCCAAAGGGGAAAGCTCACCTTATCCTGGATCCCAGTGGCTTCCACCCCTGCTGTATCTGCCAGTACCTGTCTTATTTCCCCCAAATGCTTCCGCAGATTTCTTCTGTACTTTGCAAGGCACACCATGTCACCCACACCCCTCCTCTTCCCTTTTTATATCCCATTTTTTATATCAATTTCTTATTTTACAATAAAACTTTGCTACCAAAAAAAAAAAAAAAA
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Análise do seqüenciamento
• Pesquisar cadeias abertas de leitura (ORF – open reading frames) = ATG
• Existem 6 possíveis ORFs
ATTHECATATETHERAT
5´ ATATGATATGATCGACTAGCATC 3´3´ TATACTATACTAGCTGATCGTAG 5´
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