Ribotyping is a molecular technique that uses variations in rRNA genes to identify and characterize bacteria. It involves digesting bacterial DNA with restriction enzymes, separating the fragments via gel electrophoresis, and using rRNA gene probes to visualize banding patterns specific to different bacterial strains. Ribotyping provides a reproducible method for bacterial strain differentiation and has applications in taxonomy, epidemiology, and clinical and environmental microbiology. It offers automated alternatives but remains technically demanding and most suitable for reference laboratories.

1. SEMINAR TOPIC
RIBOTYPING
PRESENTED BY :– ADITI CHANDRAKAR
MSc. 1ST SEMESTER (BIOTECHNOLOGY)
GUIDED BY :– DR. K.K. SHUKLA
SCHOOL OF STUDIES IN BIOTECHNOLOGY
PANDIT RAVISHANKAR SHUKLA UNIVERSITY

2. Content
 Introduction
 History and need
 Ribosome and its types
 Ribosomal RNA (rRNA)
 Principle of Ribotyping
 16S rRNA
 Procedure of Ribotyping
 Types of Ribotyping
 Uses of Ribotyping
 Advantage and disadvantage of Ribotyping
 Reference

3. Introduction of Ribotyping
 Ribotyping is a molecular technique for bacterial identification and
characterization that uses information from rRNA – based phylogenetic
analyses.
 The name derives from the ribosome which is part of the cellular machinery
that creates proteins.
 Ribo means RIBOSOMAL RNA (rRNA)
typing means description, identification, nomenclature
 Ribotyping is also known as rRNA gene restriction pattern analysis.
 Ribotyping can be used to identify bacteria and fungi but not viruses.

4. History
 Ribosomes were first noted in plant cells by Robinson and Brown in 1953 while
studying bean roots with electron microscope.
 Ribotyping is the most applied hybridization based RFLP (Restriction Fragment
Length Polymorphism) method.
 The original scheme called rDNA restriction pattern determination, was described
in 1986 by Grimont and Grimont.
 Ribotyping was one of the first universal genotyping technique for bacteria.
 It is considered as a relatively stable and dependable system for molecular
taxonomy.

5. Need for Ribotyping
 It is needed to explore the diversity of microbes in a particular source
as well as for tracing and monitoring the occurrence of specific
organism.
 Required for clinical diagnosis and analysis of microbial communities in
food, water and beverages.
 It is needed To provide sufficient resolution for characterization and
identification of bacteria.

6. Ribosomes and it’s types
 A ribosome is an intercellular structure made of both RNA and
protein and it is the site of protein synthesis in the cell.
 Types of Ribosomes –
1. 70S Ribosomes – found in prokaryotes. It is composed of two
subunits 50S and 30S .
2. 80S Ribosomes – found in eukaryotes. It is composed of 60S
and 40S subunits.
 Here S refers to Svedbergs unit.

8. Ribosomal RNA (rRNA)
 A ribosome is composed of RNA that is folded up in a particular way.
 This is referred as “rRNA” or “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.

9.  Typically, each ribosomal operon consists of the three genes encoding the
structural rRNA molecules, 16S, 23S, and 5S, cotranscribed as a polycistronic
operon.
 In bacterial species, the average lengths of the structural rRNA genes are:-
1. 1,522 bp(base pairs) for 16S rRNA
2. 2,971 bp for 23S rRNA and
3. 120 bp for 5S rRNA
 In prokaryotes: 23S, 5S,16S structural rRNA genes are found in ribosomal
operon .
 In eukaryotes: 28S, 5.8S, 5S, 18S structural rRNA genes are found in
ribosomal operon.

10. Organization of Ribosomal RNA
operon in prokaryotes

11. 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”.

12. What is 16S rRNA?
 16S rRNA is the RNA component of 30S subunit of a prokaryotic ribosome.
 The genes coding for it are referred as 16S rRNA and are used in reconstructing
phylogenies.
 Why 16S rRNA is used for Ribotyping?
1. RIBOSOMAL RNA and Ribosomes are seen in all cells.
2. Due to slow rate of evolution of this genes
3. It is highly conserved.
4. Sequence is lengthy enough ((1500 bp approx.)
5. Contains variable regions that can provide species - specific signature sequence.
 Because of the above features of 16S rRNA gene sequencing has been established as
“GOLD STANDARD” for identification and classification of bacterial species.

13. Procedure of
Ribotyping –
Conventional
ribotyping
Pick up a single
colony from agar plate
Extract genomic DNA
Restriction digestion
Agarose gel
electrophoresis
Southern blotting
Hybridisation
Autoradiography
Data analysis
(dendrogram)
Probe
preparation
PCR amplification of
rRNA operon
32p labelling

14. Procedure of Ribotyping

15. Ribotyping protocol for Haemophilus
influenzae strain Rd
o Step 1 - In silico survey of genomic sequence of strain Rd to search for
conserved restriction endonuclease cleavage site within the six ribosomal
operon.
o Step 2 - Choose the ideal restriction enzyme.
o Step 3 - Confirm the conservation of restriction site by all publicly available
16S and 23S rRNA of H. influenzae gene sequences.
o Step 4 - Following restriction enzyme selection the genomic DNA’s of
isolates to be ribotyped are digested electrophoresis southern
blotting Hybridization to a labelled probe autoradiography data
analysis.

16. In silico analysis of the six ribosomal operons of
genomically sequenced H. influenzae strain Rd.

17. Different Types of Ribotyping
1. CONVENTIONAL RIBOTYPING -
 Is based on restriction endonuclease cleavage of total genomic DNA
followed by electrophoretic separation, Southern blot transfer , and
hybridization of transferred DNA fragments with a radiolabeled ribosomal
operon probe.
 Following autoradiography, only those bands containing a portion of the
ribosomal operon are visualized.
 The number of fragments generated by ribotyping is a reflection of the
multiplicity of rRNA operons present in a bacterial species.

18. 2. Automated ribotyping –
 It 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.
 This would be an ideal system in the clinical microbiology
laboratory because of its speed and reliability.

19. Uses of Ribotyping
 Strain differentiation within species and implemented to 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.

20. 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.

21. 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.

22. Reference
 ROGER Y. STANIER , JOHN L INGRAHAM, MARK L. WHEELIS,
GENERAL MICROBIOLOGY (FIFTH EDITION)
 WWW.NCBI.IN
 LANSING PRESCOTT, JOHN HARLEY, AND DONALD KLEIN
MICROBIOLOGY (FIFTH EDITION)