RIBOTYPING

Ribotyping
Introduction
History
Ribosomes
Ribosomal RNA
Principle of ribotyping
16S rRNA
Procedure of ribotyping
Types of ribotyping
Use of ribotyping
Advantage and disadvantage of ribotyping
Reference

1. SEMINAR TOPIC RIBOTYPING PRESENTED BY :– ADITI CHANDRAKAR MSc . 1 ST 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 1 6S 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 L ength 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 – 70S Ribosomes – found in prokaryotes. It is composed of two subunits 50S and 30S . 80S Ribosomes – found in eukaryotes. It is composed of 60S and 40S subunits. Here S refers to Svedbergs unit.

7. 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.

8. 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:- 1,522 bp (base pairs) for 16S rRNA 2,971 bp for 23S rRNA and 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.

9. Organization of Ribosomal RNA operon in prokaryotes

10. 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 “ R ibotype ”.

11. 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 ? RIBOSOMAL RNA and Ribosomes are seen in all cells. Due to slow rate of evolution of this genes It is highly conserved. Sequence is lengthy enough ((1500 bp approx.) 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.

12. Procedure of Ribotyping – Conventional ribotyping

13. Procedure of Ribotyping

14. Ribotyping protocol for Haemophilus influenzae strain Rd Step 1 - In silico survey of genomic sequence of strain Rd to search for conserved restriction endonuclease cleavage site within the six ribosomal operon . Step 2 - Choose the ideal restriction enzyme. Step 3 - Confirm the conservation of restriction site by all publicly available 16S and 23S rRNA of H. influenzae gene sequences. 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 .

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

16. Different Types of Ribotyping   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 .

17. 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.

18. 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.

19.   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 .

20. 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.

21. 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)

22. Thankyou