Yeast Identification by DNA Sequencing in an Undergraduate Mycology Laboratory

Abstract

A yeast identification procedure based on the sequencing of the D1/D2 region of the yeast 26S ribosomal DNA was presented in an undergraduate general mycology laboratory. Extracted genomic DNA of environmental yeasts collected by the students was used to PCR-amplify the D1/D2 region of the isolates. Agarose gel electrophoresis confirmed the presence of the expected amplicon which was purified for sequencing by a spincolumn method then submitted to a commercial sequencing laboratory. The students examined the sequence of their isolates for base miscalls using Applied Biosytem’s Sequence Scan program. The sequences were then used to perform a BLAST search which compared them to the D1/D2 sequences of all validly described yeast species on file in the GenBank database. Of 37 isolates tested, 33 (89%) returned usable sequences for BLAST identification. The module required two lab periods of 1.5 and 2 hours, respectively, to complete with an enrollment of 40 students.