1887

Yeast Identification by DNA Sequencing in an Undergraduate Mycology Laboratory

    Author: William W. Safranek1
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    Affiliations: 1: Department of Molecular Biology & Microbiology, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL 32816
    AUTHOR AND ARTICLE INFORMATION AUTHOR AND ARTICLE INFORMATION
    • Published 01 May 2014
    • fn1-jmbe-15-26Kindly note, the author of this article has passed away. With permission from the author’s family, the Editor-in-Chief of has reviewed the paper on the author’s behalf.
      Supplemental materials available at http://jmbe.asm.org
    • Corresponding author : William W. Safranek
    • ©2014 Author(s). Published by the American Society for Microbiology.
    Source: J. Microbiol. Biol. Educ. May 2014 vol. 15 no. 1 26-27. doi:10.1128/jmbe.v15i1.598
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    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.

Key Concept Ranking

Agarose Gel Electrophoresis
0.54199654
Yeast DNA
0.48224297
Rose Bengal
0.4801263
0.54199654

References & Citations

1. Fell Jack W, Boekhout Teun, Fonseca Alvaro, Scorzetti Gloria, Statzell-Tallman Adele 2000 Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis Int J Syst Evol Microbiol 50 1351 1371 10.1099/00207713-50-3-1351 10843082 http://dx.doi.org/10.1099/00207713-50-3-1351
2. Guadet J, Julien J, Lafey JF, Brygoo Y 1989 Phylogeny of some Fusarium species, as determined by large subunit rRNA sequence comparison Mol. Biol. Evol 6 227 242 2622333
3. Kurtzman Cletus P, Robnett Christie J 1998 Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences Antonie Van Leeuwenhoek 73 331 371 10.1023/A:1001761008817 9850420 http://dx.doi.org/10.1023/A:1001761008817
4. Lopandic K, Zelger S, Bánszky LK, Eliskases-Lechner F, Prillinger H 2006 Identification of yeasts associated with milk products using traditional and molecular techniques Food Microbiol 23 341 350 10.1016/j.fm.2005.05.001 16943023 http://dx.doi.org/10.1016/j.fm.2005.05.001
5. Starmer William, Lachance Marc-André 2011 Yeast ecology 82 Kurtzman CP, Fell JW, Boekhout T The yeast: a taxonomic study 5th ed Elsevier Burlington, MA
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/content/journal/jmbe/10.1128/jmbe.v15i1.598
2014-05-01
2017-06-25

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.

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