12.3E: Nucleic Acid Sequencing and rRNA Analysis

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Nucleic acid sequencing and rRNA analysis consist of comparing nitrogen bases in rRNA.

Learning Objectives

Describe how the 16SrRNA gene can be used for phylogenetic studies and in medical microbiology for bacterial identification

Key Points

This method is effective in identifying general group differences of pathogens.
16S rRNA gene sequences contain hypervariable regions that can provide species -specific signature sequences useful for bacterial identification.
This method can also be fine-tuned to identify pathogens at the species level.

Key Terms

ribosomes: Large and complex molecular machine, found within all living cells, that serves as the primary site of biological protein synthesis.

Sixteen S ribosomal RNA (or 16S rRNA) is a component of the 30S small subunit of prokaryotic ribosomes. It is approximately 1.5kb (or 1500 nucleotides) in length. The genes coding for it are referred to as 16S rDNA, and are used in reconstructing phylogenies. Multiple sequences of 16S rRNA can exist within a single bacterium.

Figure: **Ribosomal RNA**: Structure and shape of the E.coli 70S ribosome. The large 50S ribosomal subunit (red) and small 30S ribosomal subunit (blue) are shown with a 200 Ångstrom (20 nm) scale bar. For the 50S subunit, the 23S (dark red) and 5S (orange red) rRNAs and the ribosomal proteins (pink) are shown. For the 30S subunit, the 16S rRNA (dark blue) and the ribosomal proteins (light blue) are shown.

The 16SrRNA 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. In addition, mitochondrial and chloroplastic rRNA are also amplified. Unfortunately, while primers can be defined to amplify this gene from single genomes, this method is not accurate enough to estimate the diversity of microbial communities from their environments. Principal limits are the lack of real universal primers; DNA amplification biases and reference database selection impact the annotation of reads.

Paradoxically, methodological denial is now a rule in published articles that use 16S rRNA gene amplicon surveys to study unknown microbial communities. In these articles, one pair of primers (although many of them are designed, and provide different results) is used to amplify a region of the 16S rRNA gene. In addition to highly conserved primer binding sites, 16S rRNA gene sequences contain hypervariable regions that can provide species-specific signature sequences useful for bacterial identification. As a result, 16S rRNA gene sequencing has become prevalent in medical microbiology as a rapid and cheap (while inaccurate) alternative to phenotypic methods of bacterial identification. Although it was originally used to identify bacteria, 16S sequencing was subsequently found to be capable of reclassifying bacteria into completely new species, or even genera. It has also been used to describe new species that have never been successfully cultured.