Ribotyping: Introduction, Ribosmes and RNA, Principle, Procedure, Diffe

Ribotyping: Introduction, Ribosmes and RNA, Principle, Procedure, Different Techniques of Ribotyping and Uses

Ribotyping is a molecular diagnostic technique. Name derives from the ribosome which is part of the cellular machinery that creates proteins. The ribosomes were first noted in plant cells by Robinson and Brown in 1953 while studying bean roots with the electron microscope. Ribotyping can be used to identify bacteria and fungi but not viruses. Ribosomes are found only in cells. Viruses have no cellular structure but are molecules with genetic material and protein only.

Introduction of Ribotyping

Ribotyping is a molecular diagnostic technique. The name derives from the ribosome which is part of the cellular machinery that creates proteins. The ribosomes were first noted in plant cells by Robinson and Brown in 1953 while studying bean roots with the electron microscope. Ribotyping can be used to identify bacteria and fungi but not viruses. Ribosomes are found only in cells. Viruses have no cellular structure but are molecules with genetic material and protein only.

Ribosomes and RNA

A ribosome is composed of RNA that is folded up in a particular way. This is referred to as “rRNA” for ribosomal RNA. DNA codes for RNA and since a wide variety of living cells create proteins, the DNA genes that code for rRNA have a lot in common, even across different species. Some parts of the (DNA) genes that code for rRNA are highly variable from one species to the next or between strains of bacteria. These variable regions can therefore be used to identify a particular strain of bacteria.

Types of Ribosomes

The ribosomes are of two basic types, the 70S and 80S ribosomes and S refer to Svedberg units. Actually, it is the sedimentation coefficient that shows how fast cell organelle sediments are in an ultra-centrifugation. Typically, each ribosomal operon consists of the three genes encoding the structural rRNA molecules, 16S, 23S, and 5S, cotranscribed as a polycistronic operon. Among bacterial species, the average lengths of the structural rRNA genes are 1,522 bp, 2,971 bp, and 120 bp, for 16S, 23S, and 5S, respectively

In brief-

In prokaryotes: 23S, 5S,16S
In eukaryotes: 28S, 5.8S, 5S, 18S

Variable Regions of rRNA Determination

DNA-cutter enzymes are used to divide the RNA only when a specific sequence occurs. If a strain of bacteria has that sequence in its rRNA, the rRNA strand will be cut at that location. The rRNA is then run on a gel so that the number and size of the pieces can be seen. rRNA that has been cut in the expected locations will appear different from rRNA that was not cut.

Principle of Ribotyping

Ribotyping is a molecular method that takes advantage of unique DNA sequences to differentiate strains of organisms. The genomic DNA is cleaved at specific sites by doing a restriction digest. This generates pieces of DNA of different lengths. Since different strains of bacteria have the specific “cut-sites” of the restriction enzymes in different places, each strain generates a unique pattern of DNA pieces.

Because there would be too many pieces if one looked at the entire genome and thus it is usually compared to the pieces of DNA from the 16S and 23S rRNA genes. After restriction digestion ( cleavage of the genome), the sample is run on an agarose gel to separate the pieces, which appear as bands. To visualize only the 16s and 23s rRNA genes, a probe that hybridizes only to those genes is added. The banding pattern of DNA fragments is known as the “ribotype”.

Procedure of Ribotyping

Convention ribotyping

Pick up a single colony from the agar plate
Extract genomic DNA
Restriction Digestion
Agarose gel electrophoresis
Southern blotting
Hybridization
Autoradiography
Data analyis (dendogram)

Probe preparation

PCR amplification of rRNA operon
32P labeling
Southern blotting
Hybridization
Autoradiography
Data analyis (dendogram)

e.g. a ribotype image showing two strains of Salmonella Newport as shown above picture.

Advantages of ribotyping

This technique allows you to differentiate different strains of bacteria in a very sensitive manner.
Ribotyping is a fully automated procedure.
The procedure involves less labor and is standardized.

Disadvantage of Ribotyping

We should choose the probe carefully so there is no cross-reactivity.
We should also choose probes so that they successfully bind to sequences.
Expensive because of the equipment used, therefore usually only performed in reference laboratories.

Uses of Ribotyping

Strain differentiation within species and implemented several bacterial species like S.aureus, E coli, P. aeruginosa,H. influenzae, B.cepacia, N. meningitidis, B. pertussis, etc.
It is also applicable for fungi.
Ribotype-based differentiation of independent isolates within a species has included taxonomic classification, epidemiological tracking, geographical distribution, and population biology and phylogeny.

Different Techniques of Ribotyping

Conventional Ribotyping
Selection of Restriction Endonuclease for Ribotyping by Sequence Analysis (In Silico)
Automated Ribotyping: Automated ribotyping was first introduced by Dupont Qualicon-1995- Riboprint pattern. This system is reproducible, convenient, and fast. A single colony of bacteria is picked up and suspended in lysing buffer and transferred to the riboprinter- in the riboprinter like conventional ribotyping.
PCR Ribotyping: Advantages of ribotyping as an identification method: Ribotyping is a fully automated procedure and the procedure involves less labor and is standardized. Disadvantages of ribotyping: Expensive because of the equipment used, therefore usually only performed in reference laboratories.

PCR Ribotyping and Endonuclease Subtyping
PCR Ribotyping Followed by Sequencing of ISR
Amplified Ribosomal DNA (rDNA) Restriction Analysis (ARDRA): An alternate variation of PCR ribotyping, amplified rRNA gene restriction analysis (ARDRA) is based on PCR amplification of the 16S rRNA gene followed by restriction digestion. Jayarao et al. (34) developed this technique to determine subspecies of Streptococcus uberis, as a means to avoid methods involving DNA hybridization or sequencing. As 16S rRNA gene sequence variation for interspecies differentiation of isolates (i.e., species determination) is well established (21, 87, 91), it is not surprising that ARDRA, even more so than PCR ribotyping, has proven useful for species differentiation rather than intraspecies epidemiological discrimination or phylogenic organization of independent isolates.
Terminal Restriction Fragment Length Polymorphism of 16S rRNA Gene
Long PCR Ribotyping-Long PCR Ribotyping -As a means to enhance the differentiating capacity of PCR ribotyping followed by restriction digestion, developed long PCR ribotyping. This technique is based on PCR amplification of the entire 16S-23S-5S ribosomal operon (∼5.5 kb) followed by restriction endonuclease digestion.
Ribosomal DNA Sequence Analysis

5. In Situ Hybridization Targeted to Detect rRNA

Clone-FISH
CARD Fish
RING (Recognition of Individual Gene) FISH
Stable Isotope-Labeled Probing of rRNA and rDNA
Peptide Nucleic Acid Probes

Key Notes

Ribosomes facilitate the synthesis of proteins in cells and the process is called translation.
Ribosomes are macromolecular machines that synthesize all cellular proteins. and ribosome biogenesis requires a huge expenditure of cellular energy, and its dysregulation can result in a number of diseases including cancer.
Ribosomes are composed of two subunits, the large and the small subunit, both of which consist of ribosomal RNA (rRNA) molecules and a variable number of ribosomal proteins.
Subunit Organization and Nomenclature of Ribosomes-They are large and complex enzymes: the simplest ribosomes from bacteria have a mass of some 2.5 million Da. All ribosomes consist of two loosely associated subunits of unequal size. In the bacterial ribosome, the large subunit is denoted the 50S, the small subunit 30S, and the entire ribosome 70S, according to their rates of sedimentation during ultracentrifugation. The bacterial small subunit consists of the 16S ribosomal (rRNA), which is approximately 1500 nucleotides, and around 20 small proteins (named S1, S2, etc.). The large subunit contains the 23S rRNA (~2900 residues), the 5S rRNA (~120 residues), and around 30 proteins (L1, L2, etc.). Eukaryotic ribosomes are even more massive, with the 60S large subunit (with 5S, 28S, and 5.8S rRNAs plus proteins) and the 40S small subunit (with 18S rRNAs plus proteins) making up the 80S ribosome.
There are two types of ribosomes, free and fixed (also known as membrane-bound). They are identical in structure but differ in locations within the cell. Free ribosomes are located in the cytosol and are able to move throughout the cell, whereas fixed ribosomes are attached to the rough endoplasmic reticulum( rER).
The 16S rRNA gene is the most conserved of the three rRNA genes, 16S rRNA gene sequencing has been established as the “gold standard” for identification and taxonomic classification of bacterial species.

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