Ribotyping is a molecular technique for bacterial identification and characterization that uses information from rRNA-based phylogenetic analyses.
It is a rapid and specific method widely used in clinical diagnostics and analysis of microbial communities in food, water, and beverages.
All bacteria have
ribosomal genes, but the exact sequence is unique to each species, serving as a genetic fingerprint. Therefore, sequencing the particular 16S gene and comparing it to a database would yield identification of the particular species.
Technique
Ribotyping involves the digestion of bacterial genomic DNA with specific
restriction enzyme
A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class ...
s. Each restriction enzyme cuts DNA at a specific nucleotide sequence, resulting in fragments of different lengths.
Those fragments are then run on a
Gel electrophoresis
Gel electrophoresis is a method for separation and analysis of biomacromolecules ( DNA, RNA, proteins, etc.) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size (IEF ...
, where they are separated according to size: the application of electrical field to the gel in which they are suspended causes the movement of DNA fragments (all negatively charged due to the presence of phosphate groups) through a matrix towards the positively charged end of the field. Small fragments move more easily and rapidly through the matrix, reaching a bigger distance from the starting position than larger fragments.
Following the separation in the gel matrix, the DNA fragments are moved onto nylon membranes and hybridized with a labelled 16S or 23S rRNA probe. This way only the fragments coding for such rRNA are visualised and can be analyzed. The pattern is then digitized and used to identify the origin of the DNA by a comparison with reference organisms in a computer database.
Conceptually, ribotyping is similar to probing restriction fragments of chromosomal DNA with
cloned probes (randomly cloned probes or probes derived from a specific coding sequence such as that of a
virulence factor
Virulence factors (preferably known as pathogenicity factors or effectors in plant science) are cellular structures, molecules and regulatory systems that enable microbial pathogens ( bacteria, viruses, fungi, and protozoa) to achieve the followi ...
).
[Grimont, F., and P. A. Grimont. 1986. Ribosomal ribonucleic acid gene restriction patterns as potential taxonomic tools. Ann. Inst. Pasteur Microbiol. 137B:165–175]
See also
*
Genotyping
Genotyping is the process of determining differences in the genetic make-up (genotype) of an individual by examining the individual's DNA sequence using biological assays and comparing it to another individual's sequence or a reference sequence. ...
References
Genetics techniques
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