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ITS/NTS PCR

Analysis of the ITS region has gained significant traction and is currently preferred to other methods for yeast identification (Schoch et al., 2012). However, in some instances the NTS region can also be used to good effect, being useful for the differentiation of Saccharomyces sensu stricto yeasts (Pulvirenti et al., 2000) and Kluyveromyces species (Nguyen et al., 2000). Analysis of these sequences typically involves a preliminary PCR reaction using specific primers (Table 11.6) to amplify the designated region, and to create a sufficient quantity of DNA, which can then be analysed by alternative methods. For analysis of the ITS region, this can include analysis for total length polymorphism, restriction fragment length polymorphism (RFLP), DNA probe hybridization, and DNA sequencing. Of these, DNA sequencing is the most precise due to the vast number of full-length ITS sequences that have been deposited in databases such as GenBank. However, for brewery troubleshooting purposes RFLP tends to be used preferentially, simply due to the speed and ease at which the technique can be conducted. RFLP is a term used to describe the analysis of DNA fragments, obtained by the use of specific enzymes that cut at pre-designated locations within a DNA sequence. Restriction enzymes, originally isolated from bacteria, cut at recognition sites within the DNA giving rise to multiple fragments that can then be separated by electrophoresis (Fig. 11.7). The application of these enzymes provides a useful means of identifying yeast species, since the enzymes utilized can be selected based on their capacity to cut at a location known to highlight polymorphisms between species and genera. Since the development of the technique, a series of studies have been performed, primarily to identify important yeast strains isolated from wine (Guil- lamon et al., 1998; Esteve-Zarzoso et al., 1999), but also from clinical sources (Trost et al., 2004; Leaw et al., 2006) and a range of industrial environments including breweries (Pham et al., 2011).

The primers used for amplification of the ITS region are known to have near universal function with ascomycete yeasts (Kurtzman and Robnett, 1991) and the majority of studies employ the restriction enzymes CfoI, HinfI and HaeIII for increasing specificity. This is primarily because they have been proven to have highly suitable restriction sites. The size of the ITS region varies between species from approximately 300 bp to 1000 bp, which does allow for some differentiation, particularly at the genus level. However, individual species can only be reliably differentiated based on either RFLP or sequence-based analysis as described above. Due to a combination of the size of the ITS fragment and the restriction enzyme cutting site, the size and number of restriction fragments yielded by RFLP analysis differs according to species (Table 11.7). It should be noted that strains belonging to the Saccharomyces sensu stricto group exhibit ITS sequences in the region of 840-880 bp in size, a feature which can be used to immediately differentiate them from non-Saccharomyces contaminants. Within the sensu stricto group there are less obvious differences that can cause issues, especially if contamination or a mix-up between production yeasts is suspected. In such instances, further identification is required. There are a large number of molecular-based techniques that can be used to differentiate yeasts to the strain level. While this list is by no means exhaustive, methods that are known to be successful include analysis of yeast transposons (Wightman et al., 1996), micro- and mini- satellites (Baleiras Couto et al., 1996; Perez et al., 2001; Schuller et al., 2004), chromosomal karyotyping (Schwartz and Cantor, 1984; Casey et al., 1990; Oakley-Gutowski et al., 1991; Boekhout et al., 1993; Casey, 1996), inter-delta sequence fingerprinting (Ness et al., 1993; Legras and Karst, 2003), mitochondrial DNA profiling (Aigle et al., 1984; Lopez et al., 2001), and analysis of single nucleotide polymorphisms (SNPs) (Ben-Ari et al., 2005; Schacherer et al., 2007, 2009).

 
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