Localização Física do BAC Clone AL 619337 no Genoma de Schistosoma mansoni Utilizando a Técnica de FISH (Fluorescence in situ Hybridization)

AUTOR(ES)
DATA DE PUBLICAÇÃO

2004

RESUMO

The Schistosoma mansoni genome physical map is under construction and large DNA fragments localization by FISH is priority for this strategy. Therefore, little information about this respect is actually available. To contribute with this initiative, a BAC clone DNA was located on the S. mansoni genome by FISH technique in this work. The BAC DNA was labeled by the biotinylated 14-dCTP incorporation in a randomly primers amplification labeling system, using the Klenow polymerase. Cells on metaphase were recovered by dissection of the intramoluscan stage, secondary sporocyst. These were fixed and used to prepare slides containing metaphase spreads. The labeled BAC DNA was hybridized to the S. mansoni chromosomes and the slides were washed several times before to reveal the hybridization signal with an Avidin-FITC conjugated. Chromosomes were counter stained with Propidium Iodide in the anti fade solution, and the slides were covered and observed in an epi-fluorescent microscopy coupled to a digital camera to capture the images. C banding was performed to help in identifying and assembling the chromosomes. We were able to establish the better infection time of the snail that yielded good amounts of metaphase spreads. The PI and FITC fluorescence were adjusted too, according to the microscopy power of resolution.The main signal was observed in the heterochromatineuchromatin transition region of the chromosome W short arm in all female metaphases observed, but nether on the chromosome Z of the male metaphases, showing that the localized BAC clone contains a female specific DNA.With this work, we hope to contribute to the S. mansoni genome physical mapping, becoming an additional place to obtain DNA localization by FISH for this important worm. After the sequencing of the genome is finished, gaining knowledge of the sequences localization is an important step. It will help to associate the physical structure of genome and the functional data of the genes for better understanding different features related to the pathology caused for this important worm.

ASSUNTO(S)

s. mansoni biologia molecular bac clones genoma mapeamento físico

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