Technical Description

Early ribotyping methods used digestion of extracted rDNA with restriction endonucleases, separation of rDNA fragments by electrophoresis, and hybridization with a cDNA probe in a Southern blotting process.[1,2] Unlike conventional restriction fragment length polymorphism (RFLP) analysis, which resolves the difference between bacterial isolates by comparison of ethidium bromide-stained DNA bands, ribotyping uses a cDNA probe to resolve differences between isolates by generating a smaller number of bands. The number of bands will depend on how many fragments the cDNA can hybridize to and therefore the number of cutting sites for the restriction endonuclease.[3,4] REs employed for ribotyping are frequent cutters such as EcoRl, BamHl, and Pvu2, but some species require less commonly used restriction endonucleases such as Pvu2.[5] DNA probes hybridize to either 16s or 23s rDNA or both. Ribotyping probes include an Escherichia coli DNA and other sequences.[6,7] The membrane used for blotting can be either nitrocellulose or nylon, and the labeling system can be a fluo-rophore, digoxigenin, or a radioisotope.

Several variations have been introduced to adapt ribotyping for wider use. A version of ribotyping based on digestion with EcoRl, an E. coli cDNA probe, and a fluorescent tracer has been automated to simplify and speed up the procedure[8](Fig. 1). In PCR ribotyping, PCR primers to highly conserved regions of the 16S-23S intergenic spacer region are used to amplify the product for subsequent length polymorphism analysis.[9]

In outline, the manual ribotyping method is as follows:

1. Bacterial DNA extraction (phenol-chloroform, gua-nidium, etc.).

2. DNA check (concentration and mini-gel).

3. Digestion with restriction endonuclease.

4. Preparatory gel for blotting.

5. Southern blot.

6. rDNA digoxigenin labeling.

7. DNA marker ladder digenin labeling.

8. Prehybridization.

9. Hybridization.

10. Wash and development.

11. Ribotype pattern recording and analysis.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

Get My Free Ebook


Post a comment