16S Ribosomal RNA Gene Sequencing PDF

Molecular structure of the 30S Subunit from Thermus thermophilus. Proteins are shown 16S Ribosomal RNA Gene Sequencing PDF blue and the single RNA strand in orange.

Författare: Sandra Tscherwizek.
In biopharmaceutical production areas the microbial
count should be kept to a minimum to ensure the
stability and cleanliness of drug products. In
general, the conventional API and VITEK methods are
used to characterize them. However, more and more
frequently, microbiologists isolate "difficult"
strains that such automated systems often fail to
identify. Those "difficult" strains are often found
in clean room areas, because the use of
disinfectants leads to a change in the metabolic
pattern and so the conventional methods provide
wrong results. The author, Sandra Tscherwizek, shows
an opportunity to identify such difficult strains
via a 16S rRNA gene sequencing method based on the
genetic level, and describes how to interpret the
outcome. Furthermore a cost-benefit calculation was
done to compare the conventional methods Vitek and
API with the sequencing method. The study shows that
an identification of microorganisms by 16S rRNA gene
sequencing leads to higher resolution power compared
to conventional biochemical methods.

Multiple sequences of the 16S rRNA gene can exist within a single bacterium. RNA, it has a structural role, acting as a scaffold defining the positions of the ribosomal proteins. The 3′ end contains the anti-Shine-Dalgarno sequence, which binds upstream to the AUG start codon on the mRNA. The 16S rRNA gene is used for phylogenetic studies as it is highly conserved between different species of bacteria and archaea. Carl Woese pioneered this use of 16S rRNA.

The most common primer pair was devised by Weisburg et al. In addition to highly conserved primer binding sites, 16S rRNA gene sequences contain hypervariable regions that can provide species-specific signature sequences useful for identification of bacteria. 30-100 base pairs long that are involved in the secondary structure of the small ribosomal subunit. While 16S hypervariable regions can vary dramatically between bacteria, the 16S gene as a whole maintains greater length homogeneity than its Eukaryotic counterpart, which can make alignments easier. While 16S hypervariable region analysis is a powerful tool for bacterial taxonomic studies, it struggles to differentiate between closely related species.

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