Ribotyping

Principles


Ribotyping is a molecular technique that takes advantage of unique DNA sequences to differentiate strains of bacteria. The genomic DNA is cut at specific sites by doing a restriction digest. This generates pieces of DNA of different lengths. Because 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, we usually compare the pieces of DNA from the 16s and 23s rRNA genes. Once the genome is cut, 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

  1. Extract the genomic DNA from your bacterial isolate
  2. Cut it with a specific restriction enzyme
  3. Run the DNA in an agarose gel by electrophoresis. DNA fragments are separated by size as they move through the gel
  4. Transfer the DNA pieces to a nylon membrane
  5. Incubate the nylon membrane with a specific enzyme-linked DNA probe (a DNA fragment that hybridizes to the genes coding for 16S and 23s rRNA).
  6. Wash the nylon membrane and add the enzyme substrate to produce a chromogenic or fluorescent signal and visualize the pieces of DNA of interest.

In the case of chromomeric signal the reaction will be seen in the nylon membrane. In the case of fluorescent reaction the nylon membrane is coupled with a photographic film and the results are seen on film.

Advantages

  • allows you to differentiate different strains of bacteria in a very sensitive manner

Disadvantages

  • Must carefully choose probe so there is no cross reactivity
  • Must carefully choose probes so that they successfully bind to sequences

Examples

http://www.cdc.gov/ncidod/eid/vol5no3/funke.htm