Comparação de métodos de extração de DNA a partir de fontes escassas
Tipo
TCC
Data de publicação
2018-12
Periódico
Citações (Scopus)
Autores
Silva, Marjorye Cristina Dias
Orientador
Pincinato, Éder de Carvalho
Título da Revista
ISSN da Revista
Título de Volume
Membros da banca
Programa
Resumo
A fim de avaliar a eficiência, nível de contaminação, toxicidade, qualidade e quantidade da extração de DNA a partir de amostras escassas, como fios de cabelo e raspado bucal, os métodos de extração com fenol-clorofórmio, extração com cloreto de lítio, Kit Wizard® Genomic DNA Purification e método de Lahiri & Nurnberger otimizado por Salazar, foram comparados. A utilização desse tipo de amostra tem aumentado, por não serem invasivas, mais facilmente obtidas e resultarem em boa quantidade e qualidade de ácido desoxirribonucleico. A análise quantitativa e a pureza do material genético extraído foi avaliada com espectrômetro NanoDrop® e fluorimetria (“QuantiFluor® dsDNA System”). Em seguida, a eficiência da extração foi avaliada a partir da reação em cadeia da polimerase (PCR), que amplificou o gene da β-globina humana, posteriormente visualizado através da análise eletroforética em gel de agarose a 2% e transiluminador de luz azul. Analisando os resultados, constatou-se que a extração pelo método de Lahiri & Nurnberger otimizado por Salazar apresentou melhor eficiência e vantagem frente aos demais métodos estudados, já que foi o único em que foi possível visualizar o gene ampliado, além de ser considerado o método mais prático, rápido e atóxico.
In order to evaluate the efficiency, level of contamination, toxicity, quality and quantity of DNA extraction from scarce samples, such as hair samples and buccal scraping, phenol-chloroform extraction methods, lithium chloride extraction, Kit Wizard® Genomic DNA Purification and Lahiri & Nurnberger's method optimized by Salazar, were compared. The use of this type of sample has increased because they are not invasive, more easily obtained and result in good quantity and quality of deoxyribonucleic acid. Quantitative analysis and purity of the extracted genetic material was evaluated with NanoDrop® spectrometer and fluorimetry ("QuantiFluor® dsDNA System"). Then, the extraction efficiency was evaluated from the polymerase chain reaction (PCR), which amplified the human β-globin gene, later visualized through the electrophoretic analysis in 2% agarose gel and blue light transilluminator. Analyzing the results, it was found that the extraction by the method of Lahiri & Nurnberger optimized by Salazar presented better efficiency and advantage over the other methods studied, since it was the only one in which it was possible to visualize the enlarged gene, besides being considered the most practical method, fast and non-toxic.
In order to evaluate the efficiency, level of contamination, toxicity, quality and quantity of DNA extraction from scarce samples, such as hair samples and buccal scraping, phenol-chloroform extraction methods, lithium chloride extraction, Kit Wizard® Genomic DNA Purification and Lahiri & Nurnberger's method optimized by Salazar, were compared. The use of this type of sample has increased because they are not invasive, more easily obtained and result in good quantity and quality of deoxyribonucleic acid. Quantitative analysis and purity of the extracted genetic material was evaluated with NanoDrop® spectrometer and fluorimetry ("QuantiFluor® dsDNA System"). Then, the extraction efficiency was evaluated from the polymerase chain reaction (PCR), which amplified the human β-globin gene, later visualized through the electrophoretic analysis in 2% agarose gel and blue light transilluminator. Analyzing the results, it was found that the extraction by the method of Lahiri & Nurnberger optimized by Salazar presented better efficiency and advantage over the other methods studied, since it was the only one in which it was possible to visualize the enlarged gene, besides being considered the most practical method, fast and non-toxic.
Descrição
Palavras-chave
DNA , extração , amostra escassa , extraction , scarce samples