Ribotyping

Ribotyping is a molecular technique for bacterial identification that uses information from rRNA-based phylogenetic analyses.[1] It is rapid and specific method widely used in clinical diagnostics and analysis of microbial communities in food, water, and beverages.[1]

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.[2]

It involves the digestion of bacterial genomic DNA with specific restriction enzymes. Each restriction enzyme cuts DNA at a specific nucleotide sequence, resulting of fragments of different lengths.[3]

Those fragments are then run on a Gel electrophoresis, 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.[4]

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.[5] The pattern is then digitized and used to identify the origin of the DNA by a comparison with reference organisms in a computer database.[1]

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).[6]

See also

References

  1. 1 2 3 Madigan, Michael T. (2012). Biology of Microorganisms. Pearson. p. 491. ISBN 978-0-321-73551-5.
  2. ↑ "Investigating Microbial Diversity: Then and Now". learn.genetics.utah.edu. Retrieved 2016-03-15.
  3. ↑ "Restriction Enzymes | ASU - Ask A Biologist". askabiologist.asu.edu. Retrieved 2016-03-13.
  4. ↑ "Gel electrophoresis". Wikipedia, the free encyclopedia.
  5. ↑ "ribotype". courses.cit.cornell.edu. Retrieved 2016-03-13.
  6. ↑ Grimont, F., and P. A. Grimont. 1986. Ribosomal ribonucleic acid gene restriction patterns as potential taxonomic tools. Ann. Inst. Pasteur Microbiol. 137B:165–175


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