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

Abstract

Determination of Optimal Conditions for PCR-RAPD Analyses in Atta sexdens rubropilosa Forel (Hymenoptera: Formicidae) ABSTRACT-PCR-RAPD has been widely used for genetic analysis in several organisms. However, due to complex interactions among the components of the PCR reaction it is unlikely that a single amplification condition can be suitable for all situations. In order to determine the optimum conditions for using PCR-RAPD in taxonomical analyses of the genus Atta, the following factors were tested: concentrations of MgCl 2 , DNA, BSA, cycling programs and methods of DNA extraction, using Fractional Factorial Design and Central Composite Design. DNA extraction methods had little influence on PCR-RAPD reactions, while the cycling programs showed the largest effect. The MgCl 2 concentration was less important than the amount of DNA used in the reaction. Using cycling program P 2 , a significant improvement in the number of DNA fragments was achieved when MgCl 2 concentration was increased to 3.0 mM and the BSA and DNA concentrations were reduced from 0.1% to 0.05% and from 2 to 1 ng/ µl, respectively. The optimum conditions for PCR-RAPD with Atta specimens obtained in this study were: 25 µl reaction with 10 mM Tris-HCl (pH 9.0), 3.0 mM MgCl 2 , 50 mM KCl, 100 µM of each dNTP, 0.2 µM of primer, 1.0 U of Taq polymerase, 25 ng template DNA (extracted by Cheung's method) and BSA 0.05%, using the amplification program which consisted of 3 min at 94ºC, 3 min at 35ºC, followed by 40 cycles of 1 min at 94ºC, 1 min at 36ºC and 2 min at 72ºC, and a 5 m final extension at 72ºC.

PCR-RAPD and PCR-RFLP polymorphisms detected in Anopheles cruzii (Diptera, Culicidae)

“…To avoid DNA contamination from parasites and/or host blood, only fasted adult F 1 progeny were used in the molecular analyses. The DNA extraction from single mosquito samples was as in the protocol of Carvalho & Vieira (2001), which was modified from Cheung et al (1993).…”

Diversidade genética de Tibraca limbativentris Stål (Hemiptera: Pentatomidae) de Santa Catarina e do Rio Grande do Sul, usando marcadores RAPD

“…Research, utilizando programa de ciclagem baseado em Carvalho & Vieira (2001): 1 ciclo a 94ºC por 2 min, 36ºC por 1 min e 72ºC por 2 min; posteriormente 40 ciclos de 94ºC por 1 min, 36ºC por 1 min e 72ºC por 2 min; e uma extensão fi nal a 72ºC por 5 min. Os produtos de cada PCR foram visualizados em corridas de eletroforese em gel de agarose 1,5% corados com 10 mg ml -1 de brometo de etídio.…”

“…Esta se baseia na identifi cação do polimorfi smo do DNA amplifi cado aleatoriamente, através da utilização de um único iniciador de seqüência arbitrária, o qual é capaz de amplifi car as seqüências de DNA incluídas entre dois sítios de anelamento (Welsh & McClelland 1990, Williams et al 1990. Apesar de ser uma técnica que usa seqüências aleatórias, muitos trabalhos têm demonstrado sua efi ciência na identifi cação de populações de insetos (Carvalho & Vieira 2001, Oliveira et al 2002e Lopes-da-Silva et al 2004.…”

Genetic diversity of the sunflower caterpillar (Chlosyne lacinia saundersii Doubleday and Hewitson) (Lepidoptera: Nymphalidae) populations determined by molecular RAPD markers

“…Although this technique results in amplification of random sequences, several authors have demonstrated its efficiency to ditinguish genotypes in insect populations (eg. Carvalho and Vieira 2001, Oliveira et al 2002, Lopes-da-Silva et al 2004. Considering that C. lacinia saundersii is an insect of distinct geographical and climatic conditions, this study aimed to evaluate the intra and interspecific genetic variation in this insect, which can contribute to the development of proper strategies for the integrated management of this pest.…”