Genetic basis of the method
To understand how ribotyping works, one must examine the organization of bacterial genetic material. The genes responsible for encoding rRNA are assembled into specific structures known as operons.
A critical property of the nucleotide sequence within these operons is its high degree of conservation. This means that these regions are extremely similar across all members of a specific bacterial species. Furthermore, these operons do not exist as a single copy in the bacterial chromosome, but are always present in multiple copies.
Principle of variability
If rRNA genes are so similar, how do we distinguish one species from another? Microorganism differentiation in ribotyping relies on three key differences that are unique to different bacterial species:
- Copy number of rRNA operons in the chromosome.
- Topography (localization) of these copies directly within the genome.
- Location of restriction sites—specific sites where enzymes cleave DNA. These sites may be located either within the operon itself or in its flanks (adjacent flanking regions).
Ribotyping technique
Today, the entire ribotyping process is fully automated, eliminating human error and accelerating results. The classic testing workflow comprises three main steps:
- Restriction. Pre-extracted bacterial DNA is treated with specialized enzymes called restriction endonucleases (restriction enzymes). Their task is to cleave the long DNA molecule into short fragments at strictly defined locations (restriction sites).
- Hybridization. The resulting DNA fragments undergo molecular hybridization with a pre-prepared, labeled rRNA of the species we intend to detect.
- Profiling. Binding produces a unique fragment pattern (profile) that is characteristic exclusively of a specific species or even an individual bacterial strain.
Transcription-mediated rRNA amplification
This related method is used primarily when it is necessary to diagnose mixed infections. Its essence lies in detecting specific bacterial rRNA through artificial accumulation followed by hybridization.
The process takes place in automated analyzers, allowing the simultaneous detection of rRNA from several bacterial species at once. The study proceeds in three stages:
- Amplification. A massive pool (numerous copies) of rRNA is synthesized on the pre-extracted DNA template. The key enzyme at this stage is DNA-dependent RNA polymerase.
- Hybridization. The massively accumulated rRNA is bound to complementary species-specific oligonucleotides (probes). To make the result visible, these probes are obligatorily labeled with fluorophores or enzymes.
- Detection. Hybridization results are read instrumentally using methods such as densitometry or enzyme-linked immunosorbent assay (ELISA).
When diagnosing mixed infections, the amplified rRNA pool is simply divided into several separate samples. Each sample receives its own labeled oligonucleotide that is strictly specific to one particular pathogen.
Briefly: answers to topical questions
What methods are used to separate DNA fragments after restriction prior to hybridization?
Separation of DNA fragments after restriction is performed using gel electrophoresis.
- Agarose gel electrophoresis is a method in which the migration rate of fragments depends on their size. Smaller fragments move faster and travel a greater distance, while larger ones move more slowly.
DNA molecules migrate from the negative charge (wells located at the top) downward toward the positive pole. As a result, shorter fragments end up lower on the electropherogram.
What are the main differences between the ribotyping method and classical PCR?
The main differences lie in the principles of detecting genetic material and the reaction stages.
| Characteristic | Ribotyping method | Classical PCR |
|---|---|---|
| Basis of the method | Analysis of genetic differences in the arrangement of rRNA genes | Amplification of specific DNA regions |
| Key steps | DNA digestion with restriction enzymes and hybridization with labeled rRNA | PCR amplification of specific regions |
How is the unique fragment profile (pattern) physically formed and visualized during ribotyping?
The unique fragment profile in ribotyping is formed as follows:
- isolated DNA is treated with restriction enzymes, which cleave the DNA into fragments;
- the resulting fragments undergo molecular hybridization with labeled rRNA of the corresponding species;
- this yields a unique fragment profile characteristic of a specific species or strain.
The provided sources indicate that the ribotyping process is fully automated, but the specific method of profile visualization is not described.
Class and exam questions
What is the primary application of the ribotyping method?
The method is used for precise identification of bacterial species, as well as for monitoring (tracking) various bacterial strains.
What are restriction sites?
These are specific regions in the DNA molecule that are recognized and cleaved by special enzymes—restriction endonucleases.
Which enzyme is key in rRNA amplification?
The main enzyme at the amplification (accumulation) stage of the rRNA pool is DNA-dependent RNA polymerase.
How is the rRNA amplification method adapted for mixed infections?
The resulting rRNA pool is divided into several samples, and a unique labeled probe (oligonucleotide) specific for detecting a particular pathogen from a mixed infection is added to each.
What else to study on this topic
- Structure and functions of rRNA operons in prokaryotes
- Mechanism of action of bacterial restriction enzymes
- Principles of molecular hybridization
- Instrumental detection: densitometry and ELISA
- Application of fluorophores in molecular biology
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