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%.Fundação para a Ciência e a Tecnologia (FCT
