PCR differentiation of commercial yeast strains using intron splice site primers

Department of Plant Science, Waite Agricultural Research Institute, University of Adelaide, Australia.
Applied and Environmental Microbiology (Impact Factor: 3.67). 01/1997; 62(12):4514-20.
Source: PubMed


The increased use of pure starter cultures in the wine industry has made it necessary to develop a rapid and simple identification system for yeast strains. A method based upon the PCR using oligonucleotide primers that are complementary to intron splice sites has been developed. Since most introns are not essential for gene function, introns have evolved with minimal constraint. By targeting these highly variable sequences, the PCR has proved to be very effective in uncovering polymorphisms in commercial yeast strains. The speed of the method and the ability to analyze many samples in a single day permit the monitoring of specific yeast strains during fermentations. Furthermore, the simplicity of the technique, which does not require the isolation of DNA, makes it accessible to industrial laboratories that have limited molecular expertise and resources.

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Available from: Peter Langridge
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    • "In the last years, studies have focused on strain differentiation using molecular methods (recently reviewed by Beh et al., 2006) such as karyotyping (Carle and Olson, 1985; Blondin and Vezinhet, 1988) and restriction fragment length polymorphism (RFLP) analysis of mDNA (Querol et al., 1992), as well as PCR-based methods including internal transcribed sequence (ITS) of ribosomal DNA sequencing (Montrocher et al., 1998), insertion site polymorphism of delta elements (Cameron et al., 1979; Ness et al., 1993), amplified fragment length polymorphism (AFLP) (De Barros Lopes et al., 1999), random amplified polymorphic DNA (RAPD) (Baleiras-Couto et al., 1996), intron splice sequence amplification (De Barros Lopes et al., 1996) and PCR of intron of mitochondrial genes (Lo´pez et al., 2003). These methods represent an enhancement with respect to classical physiological tests in terms of resolution and processing time, but some of them have limits because they are hardly applicable for routine analysis or, when they are easy to implement, they insufficiently discriminate at the strain level. "

    Full-text · Conference Paper · Sep 2014
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    • "The yeasts were preliminarily grouped according to various characteristics, including their colony morphology and standard tests for growth on different carbon and nitrogen sources (Kurtzman et al. 2011). Physiology-based groupings were confirmed by PCR fingerprinting using the Intron Splice Site primer EI-1 (5 ´ CTGGCTTGGTGTATGT) (De Barros et al. 1996). Yeast strains with identical DNA banding patterns were grouped and putatively considered to belong to the same species (Rosa et al. 2007; Cadete et al. 2012). "
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    • "The S. sensu stricto complex is not readily distinguished from other members of the Saccharomyces group using classical microbiological methods because of its frequent exchange of genetic material, high genetic variability and the limited ribosomal RNA divergence (Mortimer et al., 1994; Wolfe and Shields, 1997; Groth et al., 2000; Monch and Stahl, 2000; Puig et al., 2000). Thus, various techniques have been developed for identifying yeast species including mitochondrial DNA restriction profiling (Aigle et al., 1984), DNA–DNA reassociation (Martini and Martini, 1987), pulsed-field electrophoresis karyotyping (Blondin and Vezinhet, 1988), mitochondrial DNA restriction endonuclease profiling (Guillamon et al., 1994), randomly amplified polymorphic DNAs (RAPDs) (Molnar et al., 1995), PCR amplification using primers based on intron splicing sites (De Barros Lopes et al., 1996), the analysis of the 18S rRNA gene (James et al., 1997), the analysis of the internal transcribed spacers (ITS) region (Montrocher et al., 1998), the analysis of the D1/D2 region of the 26S rRNA gene (Kurtzman and Robnett, 1998), amplified fragment length polymorphism (AFLP) based analysis (Azumi and Goto-Yamamoto, 2001), and microsatellite-based techniques (Hennequin et al., 2001). More recently, the development of DNA microarrays has allowed the study of genetic diversity and variation at a genomic level in Fig. 1. "
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