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Limitations on the use of polymerase chain reaction – restriction fragment length polymorphism analysis of the rDNA NTS2 region for the taxonomic classification of the species Saccharomyces cerevisiae

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Abstract:

Different molecular techniques were tested to determine which was the most effective in the identification of Saccharomyces cerevisiae strains. In particular, polymerase chain reaction – restriction fragment length polymorphism (PCR–RFLP) analysis of the internal transcribed spacer (ITS) regions and the nontranscribed spacer 2 (NTS2) region, sequencing of the D1/D2 domain, and electrophoretic karyotyping were applied to 123 yeast strains isolated from different sourdoughs and tentatively attributed to the species S. cerevisiae. All of the strains tested showed an identical PCR–RFLP pattern for the ITS regions, an identical nucleotide sequence of the D1/D2 domain, and the typical electrophoretic karyo type of S. cerevisiae. In contrast, 14 out of the 123 strains tested showed some polymorphism with BanI restriction analysis of the NTS2 region. Our results indicate that while the sequencing of the D1/D2 domain, the PCR–RFLP analysis of the ITS regions, and the electrophoretic karyotype can be employed successfully to identify S. cere visiae strains, PCR–RFLP analysis of the NTS2 region does not allow a consistent and accurate grouping for S. cere visiae strains. The fact that the NTS2 region of a small number of strains (8.78% of the total strains tested) is different from that of the other S. cerevisiae strains confirms that molecular methods should always be tested on a great number of strains.Key words: ribosomal DNA, Saccharomyces cerevisiae, yeast identification.

Différentes techniques moléculaires ont été utilisées afin de déterminer laquelle était convenable pour l'identification de souches de Saccharomyces cerevisiae. Nous avons examiné 123 souches de levures isolées de différents levains et d'abord attribuées à l'espèce S. cerevisiae. Toutes les souches ont été testées en utilisant les techniques suivantes: le PCR–RFLP de régions ITS, le PCR–RFLP de la région de l'espaceur non transcrit 2 (NTS2), le séquençage du domaine D1/D2 et le caryotypage électrophorétique. Toute les souches ont donné le même motif de restriction de S. cerevisiae d'après le PCR–RFLP des régions ITS, les séquences de nucléotides du domaine D1/D2 étaient identiques et des caryotypes électrophorétiques typiques de l'espèce S. cerevisiae furent obtenus. En revanche, l'analyse de restriction de la région NTS2 avec BanI a révélé un certain polymorphisme chez 14 des 123 souches. Nos résultats indiquent que bien que le séquençage du domaine D1/D2, le PCR–RFLP des régions ITS et le caryotype électropho r étique sont utiles pour l'identification de souches de S. cerevisiae, la polymorphie de la région NTS2 par PCR–RFLP ne permet pas de regroupement adéquat de souches de S. cerevisiae. Le fait que les régions NTS2 d'un nombre limité de souches (8,78 % du nombre total de souches analysées) sont différentes des autres souches de S. cerevisiae con firme que ces méthodes moléculaires doivent toujours être testées sur un grand nombre de souches.Mots clés : ADN ribosomal, Saccharomyces cerevisiae, identification des levures.[Traduit par la Rédaction]

Document Type: Research Article

Publication date: September 1, 2005

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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