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Analysis of yeast population dynamics by Fluorescence in situ hybridisation (FISH) (CAT#: STEM-MB-1189-WXH)

Introduction

In wine production, natural grape juice fermentation is carried out by a succession of different yeast populations. The early stages of the alcoholic fermentation are generally dominated by apiculate yeasts belonging to the genus Hanseniaspora (Kloeckera), as well as by other non-Saccharomyces yeasts like Candida stellata and Metschnikowia pulcherrima. Less frequently, species assigned to the genera Pichia and Kluyveromyces are also found. As the fermentation progresses, the non-Saccharomyces species successively die off, leaving S.cerevisiae to dominate and complete the fermentation. Although some non-Saccharomyces wine species are often cited as important to the wine flavour and quality, the impact of these yeasts on the final product is still a controversial issue.




Principle

FISH uses fluorescent probes with complementary base sequences to locate the presence or absence of specific portions of DNA on chromosomes. The probe and target DNA must be denatured with heat or chemicals to break hydrogen bonds in the DNA and to allow hybridisation to occur once the two samples are mixed. The fluorescent probes form new hydrogen bonds with their complementary base pairs on the DNA, and these can then be detected via microscopy.

Applications

Detect and localize the presence or absence of specific DNA sequences on chromosomes.
Detect and localize specific RNA targets (mRNA, lncRNA and miRNA) in cells, circulating tumor cells, and tissue samples.

Procedure

1. Sample preparation
2. Co-denaturation and hybridization
3. Probe detection
4. Wash off of unbound probe
5. Analysis by flow cytometer/fluorescence microscopy

Materials

• Flow cytometer
• Fluorescence microscopy