Genotyping of Saccharomyces cerevisiae strains by interdelta sequence typing using automated microfluidics

• Autor(a) principal: Duarte, Ricardo Franco
• Data de Publicação: 2011
• Outros Autores: Gomes, Ana Catarina, Santos, Manuel A. S., Sousa, Bruno, Schuller, Dorit Elisabeth, Mendes, Inês
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/1822/13514
• topic: Capillary electrophoresis; Interdelta sequences; Non-parametric methods; Saccharomyces cerevisiae; Science & Technology

Amplification of genomic sequences flanked by delta elements of retrotransposons TY1 and TY2 is a reliable method for characterization of Saccharomyces cerevisiae strains. The aim of this study is to evaluate the usefulness of microfluidic electrophoresis (Caliper LabChip®) to assess the factors that affect interlaboratory reproducibility of interdelta sequence typing for S. cerevisiae strain delimitation. We carried out experiments in two laboratories, using varying combinations of Taq DNA polymerases and thermal cyclers. The reproducibility of the technique is evaluated using non-parametric statistical tests and we show that the source of Taq DNA polymerase and technical differences between laboratories have the highest impact on reproducibility, whereas thermal cyclers have little impact. We also show that the comparative analysis of interdelta patterns is more reliable when fragment sizes are compared, than when absolute and relative DNA concentrations of each band are considered. Interdelta analysis based on a smaller fraction of bands with intermediate sizes between 100 and 1000 bp yield the highest reproducibility.