Determinação das Condições Ótimas para Análises de PCR-RAPD em Atta sexdens rubropilosa Forel (Hymenoptera: Formicidae)

Neotropical Entomology (Impact Factor: 0.85). 01/2001; DOI: 10.1590/S1519-566X2001000400013
Source: DOAJ

ABSTRACT A técnica PCR-RAPD tem sido amplamente empregada em estudos genéticos de populações de vários organismos. Entretanto, devido às interações entre os componentes da reação de PCR é improvável que uma única condição de amplificação possa ser adequada para todas as situações. Com o objetivo de determinar as condições ótimas para a utilização da técnica PCR-RAPD em análises taxonômicas do gênero Atta, foram analisados os fatores: concentrações de MgCl2, DNA, BSA, programas de temperaturas e métodos de extração de DNA, utilizando-se os delineamentos Fatorial Fracionado e o Central Composto. Dentre os fatores avaliados, o método de extração de DNA teve pouca influência na reação, enquanto que o programa de temperatura apresentou o maior efeito. Embora a concentração de MgCl2 seja importante para obtenção de um padrão exato de bandas, seu efeito foi menor do que a quantidade de DNA. Com o aumento da concentração de MgCl2 para 3,0 mM e com a diminuição da concentração de BSA de 0,1% para 0,05% e da quantidade de DNA de 2 para 1 ng/mil ocorreu um aumento significativo no número de fragmentos amplificados, sendo esse efeito observado com maior evidência no programa P2. As condições ótimas estabelecidas para as reações PCR-RAPD foram: 25 mil de solução contendo 10 mM Tris-HCl (pH 9,0), 50 mM KCl, 3,0 mM MgCl2, 100 miM de cada dNTP, 0,2 miM de primer, 1,0 U de Taq polimerase, 25 ng DNA (extraído pelo método Cheung) e BSA 0,05%, utilizando-se o programa de amplificação: 3 min. a 94ºC, 3 min. a 35ºC, seguidos de 40 ciclos de 1 min. a 94ºC, 1 min. a 36ºC e 2 min. a 72ºC, com extensão final de 5 min. a 72ºC.

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