A new high-throughput method for the rapid extraction of yeast DNA

• Autor(es): Vieira, E. ; Neves, J. Drumonde ; Lima, Maria Teresa ; Araújo, Isabel M. ; Casal, Margarida ; Schuller, Dorit
• Data: 2011
• Identificador Persistente: http://hdl.handle.net/1822/13502
• Origem: RepositóriUM - Universidade do Minho
• Assunto(s): DNA extraction; High-throughput; Yeast; Saccharomyces cerevisiae
• Tipo de Documento: Documento de conferência
• Idioma: Inglês

Descrição

Ecological, evolutionary and population genetic studies of yeasts require the processing of a high numbers of isolates. The current molecular methods for yeast identification and characterization require time-consuming and labor intensive DNA extraction protocols. Within this research, we aimed to develop a high-throughput method for yeast DNA extraction. Total yeast DNA extraction was based on a previously described standard miniprep method (Lopez et al., 2001; Schuller et al., 2004). Several steps were simplified or skipped. Microplates containing 30, 50, 100 or 150 μL yeast cell suspensions per well were used instead of microtubes. All reagents volumes were reduced in proportion to the volumes of cell suspensions. The most suitable yeast cell suspensions were 100 and 150 μL. The quality of the obtained DNA was not affected by the modifications that were introduced. The final DNA concentration obtained with the modified protocol was in the range of 20-50 ng/μL, being most suitable for the usual DNA amplification protocols. DNA concentrations using the conventional method were much higher (800 ng/μL) and required time-consuming dilutions prior to PCR amplifications. To validate the modified method, a group of 12 yeast species and 12 Saccharomyces cerevisiae strains were processed. DNA restriction fragment length polymorphism (RFLP) of ITS sequences and interdelta sequence analysis were performed using the DNA obtained by the conventional and the herein presented method and the results were identical. This high-throughput method for yeast DNA extraction allows the processing of 1600 yeast isolates per day, which is a ten-fold increase compared to the conventional method. The required reagents volumes were reduced by 90%.