Suppression-subtractive hybridization: A rapid and inexpensive detection methodology for upregulated Perkinsus olseni genes.


Ascenso RMT, Leite RB, Afonso R, Cancela ML*

A cDNA forward subtraction library was constructed from the mollusc parasite Perkinsus olseni exposed to hemolymph from its natural host, the clam Ruditapes decussatus, and two different methodologies were used to unravel different non-redundant contigs. Our results demonstrated that screening of the non-enriched direct cDNA subtractive library (Dfsl) was the most efficient and least time- consuming method. It facilitated the identification of genes belonging to 25 different classes of molecular functions out of the 96 clones analyzed. In contrast, only 6 different classes from 204 sequenced clones were identified from the enriched library (efMOSl). It was concluded that the Dfsl cDNA subtractive library resulted in a larger pool of diversified gene hits that were obtained in a shorter time and with less technically- demanding methodology when compared to the efMOSl approach, thus demonstrating its significance and usefulness when time and/or resources are limited

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