Identification of yeast strains using the polymerase chain reaction
TL;DR: A novel strategy has been developed for identifying yeast strain employing polymerase chain reaction technology using customised oligonucleotides, some regions of the yeast genome between δ elements are amplified to give an 'amplified' sequence polymorphisml characteristic of the strains.
Abstract: Commonly used techniques for the identification of industrial yeast strains are usually time-consuming and cumbersome. Moreover, some of these methods may give ambiguous results. A novel strategy has been developed for identifying yeast strain employing polymerase chain reaction technology. Using customised oligonucleotides, some regions of the yeast genome between δ elements are amplified to give an ‘amplified’ sequence polymorphisml (Skolnick and Wallace 1988) characteristic of the strains. With this technique it is possible to identify individual strains of Saccharomyces cerevisiae.
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TL;DR: PCR-DGGE of a portion of the 26S rRNA gene was shown to distinguish most yeast genera associated with the production of wine and represents an attractive alternative to traditional plating schemes for analysis of the microbial successions inherent in the fermentation of wine.
Abstract: We present a method to directly characterize the yeast diversity present in wine fermentations by employing denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 26S ribosomal RNA (rRNA) genes. PCR-DGGE of a portion of the 26S rRNA gene was shown to distinguish most yeast genera associated with the production of wine. With this method the microbial dynamics in several model wine fermentations were profiled. PCR-DGGE provided a qualitative assessment of the yeast diversity in these fermentations accurately identifying populations as low as 1000 cells ml−1. PCR-DGGE represents an attractive alternative to traditional plating schemes for analysis of the microbial successions inherent in the fermentation of wine.
439 citations
01 Jan 1979
TL;DR: In this paper, Dispersed repetitive DNA sequences from yeast (Saccharomyces cerevisiae) nuclear DNA have been isolated as molecular hybrids in lambdagt and showed marked alterations in the size of the restriction fragments containing these repetitive DNAs.
Abstract: Dispersed repetitive DNA sequences from yeast (Saccharomyces cerevisiae) nuclear DNA have been isolated as molecular hybrids in lambdagt. Related S. cerevisiae strains show marked alterations in the size of the restriction fragments containing these repetitive DNAs. "Ty1" is one such family of repeated sequences in yeast and consists of a 5.6 kilobase (kb) sequence including a noninverted 0.25 kb sequence of another repetitious family, "delta", on each end. There are about 35 copies of Ty1 and at least 100 copies of delta (not always associated with Ty1) in the haploid genome. A few Ty1 elements are tandem and/or circular, but most are disperse and show (along with delta) some sequence divergence between repeat units. Sequence alterations involving Ty1 elements have been found during the continual propagation of a single yeast clone over the course of a month. One region with a large number of delta sequences (SUP4) also shows a high frequency of sequence alterations when different strains are compared. One of the differences between two such strains involves the presence or absence of a Ty1 element. The novel joint is at one inverted pair of delta sequences.
385 citations
TL;DR: The analysis of 53 commercial and laboratory Saccharomyces cerevisiae yeast strains showed a clear improvement of interdelta analysis using the newly designed primers.
Abstract: A new primer pair (delta12–delta21) for polymerase chain reaction-based yeast typing was designed using the yeast genome sequence. The specificity of this primer pair was checked by the comparison of the electrophoresis pattern with a virtual profile calculated from Blast data. The analysis of 53 commercial and laboratory Saccharomyces cerevisiae yeast strains showed a clear improvement of interdelta analysis using the newly designed primers.
317 citations
TL;DR: In the present work Lachancea (Kluyveromyces) thermotolerans and Saccharomyces cerevisiae were evaluated in simultaneous and sequential fermentation with the aim to enhance acidity and improve the quality of wine.
Abstract: In the last few years there is an increasing interest on the use of mixed fermentation of Saccharomyces and non- Saccharomyces wine yeasts for inoculation of wine fermentations to enhance the quality and improve complexity of wines. In the present work Lachancea ( Kluyveromyces ) thermotolerans and Saccharomyces cerevisiae were evaluated in simultaneous and sequential fermentation with the aim to enhance acidity and improve the quality of wine. In this specific pairing of yeast strains in mixed fermentations ( S. cerevisiae EC1118 and L. thermotolerans 101), this non- Saccharomyces yeast showed a high level of competitiveness. Nevertheless the S. cerevisiae strain dominated the fermentation over the spontaneous S. cerevisiae strains also under the industrial fermentation conditions. The different condition tested (modalities of inoculum, temperature of fermentation, different grape juice) influenced the specific interactions and the fermentation behaviour of the co-culture of S. cerevisiae and L. thermotolerans . However, some metabolic behaviours such as pH reduction and enhancement of 2-phenylethanol and glycerol, were shown here under all of the conditions tested. The specific chemical profiles of these wines were confirmed by the sensory analysis test, which expressed these results at the tasting level as significant increases in the spicy notes and in terms of total acidity increases.
316 citations
TL;DR: Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function.
Abstract: Over the course of billions of years, natural evolution has produced new proteins and adapted existing ones so that they work better. Scientists have learned how to use the principles that underlie evolution to similarly engineer proteins in the laboratory. This process, known as directed evolution, is a powerful tool for improving how proteins function. Directed evolution normally involves mutating the gene that encodes the protein of interest, selecting the genes that produce the most promising proteins for another round of mutation, and repeating the process until the desired protein function is achieved. In the first step of directed evolution, a gene is usually mutated randomly in order to create a large ‘library’ of different forms of the gene. These are joined to circular pieces of DNA known as plasmids that can replicate themselves inside cells. However, the number of plasmids than can be taken up differs from cell to cell. This complicates experiments, and the ideal directed evolution experiment would have the same number of plasmids, or target genes, being delivered into each cell. Ryan et al. have developed a new method for performing directed evolution experiments that uses a recently developed technique called the CRISPR-Cas9 system. This can make direct changes to a DNA strand such as inserting or deleting specific sequences that code for proteins. Ryan et al. used the CRISPR-Cas9 system to create multiple DNA breaks simultaneously across the genome of yeast cells, and joined ‘barcoded’ DNA or DNA for intact genes to these breaks. This avoids the need to use plasmids to introduce foreign DNA into cells. Ryan et al. have named this method the Multiplex CRISPR (or CRISPRm) system. Having established CRISPRm, Ryan et al. tested whether it could be used to engineer improved proteins by attempting to modify a transporter protein called CDT-1. This protein transports the sugar cellobiose into yeast cells, where it can be converted into alcohol by fermentation. This is important for making biofuel from plants. After just one round of directed evolution using CRISPRm, Ryan et al. successfully isolated a form of the CDT-1 protein that increased the rate of fermentation over 10-fold; hence this CDT-1 variant could be used to increase biofuel production. In the future, it will be important to implement multiple selection rounds with CRISPRm, and to test how large the DNA libraries can be for directed evolution. In time, CRISPRm could find use in evolving and engineering different combinations of genes, metabolic pathways, and possibly entire genomes.
316 citations
Cites background or methods from "Identification of yeast strains usi..."
...cerevisiae strain ATCC4124 that has superior tolerance and productivity phenotypes (Ness et al., 1993)....
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...We also tested efficacy of our approach in the polyploid industrial S. cerevisiae strain ATCC4124 that has superior tolerance and productivity phenotypes (Ness et al., 1993)....
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References
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TL;DR: A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction, which significantly improves the specificity, yield, sensitivity, and length of products that can be amplified.
Abstract: A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.
17,663 citations
TL;DR: Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia, using primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase of target DNA copies.
Abstract: Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia. The first involves the primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase (220,000 times) of target DNA copies. In the second technique, the presence of the beta A and beta S alleles is determined by restriction endonuclease digestion of an end-labeled oligonucleotide probe hybridized in solution to the amplified beta-globin sequences. The beta-globin genotype can be determined in less than 1 day on samples containing significantly less than 1 microgram of genomic DNA.
9,107 citations
TL;DR: The generality of the arbitrarily primed PCR method is demonstrated by application to twenty four strains from five species of Staphylococcus, eleven strains of Streptococcus pyogenes and three varieties of Oryza sativa.
Abstract: Simple and reproducible fingerprints of complex genomes can be generated using single arbitrarily chosen primers and the polymerase chain reaction (PCR). No prior sequence information is required. The method, arbitrarily primed PCR (AP-PCR), involves two cycles of low stringency amplification followed by PCR at higher stringency. We show that strains can be distinguished by comparing polymorphisms in genomic fingerprints. The generality of the method is demonstrated by application to twenty four strains from five species of Staphylococcus, eleven strains of Streptococcus pyogenes and three varieties of Oryza sativa (rice).
5,472 citations
TL;DR: This pulsed field gradient gel electrophoresis fractionates intact S. cerevisiae chromosomal DNA, producing a molecular karyotype that greatly facilitates the assignment of genes to yeast chromosomes.
Abstract: A new type of gel electrophoresis separates DNA molecules up to 2000 kb with resolutions exceeding the logarithmic molecular weight dependence of conventional electrophoresis. The technique uses 1.5% agarose, 10 to 20 micrograms of DNA per well, and low ionic strength buffers. It employs alternately pulsed, perpendicularly oriented electrical fields, at least one of which is inhomogeneous. The duration of the applied electrical pulses is varied from 1 sec to 90 sec to achieve optimal separations for DNAs with sizes from 30 to 2000 kb. This pulsed field gradient gel electrophoresis fractionates intact S. cerevisiae chromosomal DNA, producing a molecular karyotype that greatly facilitates the assignment of genes to yeast chromosomes. Each yeast chromosome consists of a single piece of DNA; the chromosome sizes are consistent with the genetic linkage map. We also describe a general method for preparing spheroplasts, and cell lysates, without significant chromosomal DNA breakage.
2,654 citations
TL;DR: To release plasmid DNA for the transformation of Escherichia coli, cells are subjected to vortex mixing in the presence of acid-washed glass beads, Triton X-100, sodium dodecyl sulfate, phenol and chloroform.
Abstract: A procedure for the rapid isolation of DNA from the yeast Saccharomyces cerevisiae is described. To release plasmid DNA for the transformation of Escherichia coli, cells are subjected to vortex mixing in the presence of acid-washed glass beads, Triton X-100, sodium dodecyl sulfate, phenol and chloroform. Centrifugation of this mixture separates the DNA from cellular debris. E. coli can be efficiently transformed with plasmid present in the aqueous layer without further purification of the plasmid DNA. This procedure also releases chromosomal DNA. Following two ethanol precipitations, the chromosomal DNA can be digested by restriction endonucleases and analysed by Southern blot analysis.
2,483 citations