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Desenvolvimento de um procedimento laboratorial de Next Generation Sequencing para ADN de sequenciação inviável pelo método convencional de Sanger
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Resumo(s)
O aparecimento da engenharia genética contribuiu significativamente e de forma muito
concreta para a manipulação do genoma de organismos vivos, levando a importantes
descobertas sobre a estrutura do ácido desoxirribonucleico e da descodificação do código
genético.
Com base nestes avanços e estudos revolucionários posteriormente realizados, é atualmente
possível a realização de estudos de sequenciação de ADN em que, com base em métodos
bioquímicos se determina a ordem exata das bases nitrogenadas de uma amostra
desconhecida.
Um dos objetivos deste trabalho foi implementar um protocolo alternativo que permita
colmatar as limitações da técnica de Sanger. Para a elaboração deste protocolo, o trabalho
englobou a preparação da biblioteca de ADN, foram realizadas otimizações de processos como
a fragmentação, amplificação e Size Selection. Para o processo de sequenciação, recorremos à
metodologia de sequenciação de segunda geração da Ion Torrent PGM™ System por este ter a
capacidade de suportar biliões de reações de sequenciação ao mesmo tempo, a fim de
produzir a sequência de bases de uma amostra de interesse. Desta forma tentou-se dar
resposta aos casos que não foram possíveis de sequenciar pela tecnologia tradicional de
Sanger.
Com o resultado deste trabalho demos conta de várias limitações mais típicos que o método
de Sanger apresenta e, recorrendo à tecnologia de Next Generation Sequencing, conseguimos
identificar essencialmente problemas de contaminação, amostras de ADN sem sítio específico
de ligação do primer à sequência de ADN e de formação de estruturas secundárias.
A implementação do protocolo para o uso do Ion Torrent PGM™ System, permitiu assim
identificar e estudar as causas de difícil sequenciação de amostras de ADN inviáveis.
Genetic engineering, as a tool that allow us the manipulation of an organism's genome, has contributed significantly for seminal scientific advances that span from the discovery of DNA structure to the sequencing of human genome or the cloning of entire mammal organisms. Based on consolidated technical advances and constantly emerging novel technologies, it is now possible to perform DNA sequencing studies with high efficacy and resolution. An important goal of this study consisted on the implementation of a novel protocol that could overcome the limitations associated with the Sanger sequencing. For elaboration of the protocol, the work includes the preparation of DNA libraries. In order to elaborate this alternative protocol, optimizations were performed on the following processes: Fragmentation, amplification and Size Selection of the samples. In this study we used Ion Torrent PGM™ System as a platform for Next Generation Sequencing (NGS). This technology has the ability to support billions of sequencing reactions at the same time, which allowed us to efficiently sequence DNA samples that were not possible to sequence by Sanger. With the completion of this study, and taking advantage of the NGS techniques, we were able to detect the more frequent limitations associated with Sanger sequencing. Specifically, we could assess by NGS that the most common causes of unsuccessful results in Sanger were associated with DNA contamination, with the lack of specificity (or complementarity) of the primers used, or with the formation of secondary structures such as hair-spin. The implementation of protocol for Ion Torrent PGM™ System proved to be a valuable tool to identify and/or evaluate the causes that compromise the sequencing of DNA samples considered to be of low viability.
Genetic engineering, as a tool that allow us the manipulation of an organism's genome, has contributed significantly for seminal scientific advances that span from the discovery of DNA structure to the sequencing of human genome or the cloning of entire mammal organisms. Based on consolidated technical advances and constantly emerging novel technologies, it is now possible to perform DNA sequencing studies with high efficacy and resolution. An important goal of this study consisted on the implementation of a novel protocol that could overcome the limitations associated with the Sanger sequencing. For elaboration of the protocol, the work includes the preparation of DNA libraries. In order to elaborate this alternative protocol, optimizations were performed on the following processes: Fragmentation, amplification and Size Selection of the samples. In this study we used Ion Torrent PGM™ System as a platform for Next Generation Sequencing (NGS). This technology has the ability to support billions of sequencing reactions at the same time, which allowed us to efficiently sequence DNA samples that were not possible to sequence by Sanger. With the completion of this study, and taking advantage of the NGS techniques, we were able to detect the more frequent limitations associated with Sanger sequencing. Specifically, we could assess by NGS that the most common causes of unsuccessful results in Sanger were associated with DNA contamination, with the lack of specificity (or complementarity) of the primers used, or with the formation of secondary structures such as hair-spin. The implementation of protocol for Ion Torrent PGM™ System proved to be a valuable tool to identify and/or evaluate the causes that compromise the sequencing of DNA samples considered to be of low viability.
Descrição
Palavras-chave
Engenharia genética Sanger Tecnologias de Sequenciação Next Generation Sequencing Ion Torrent Genetic engineering Sequencing Technologies