Physicochemical Basis of the Method
The foundation of the molecular hybridization method is the unique physicochemical property of the DNA molecule to undergo reversible structural changes in response to environmental temperature fluctuations.
Key processes occurring in DNA:
- Denaturation: The unwinding of the strands. It is triggered by heating the medium to $90^\circ\text{C}$ in an alkaline environment. As a result, the double-stranded structure breaks down into individual single strands.
- Renaturation: The restoration of the original double-stranded helix. For the strands to reconnect, the temperature must be lowered by $10^\circ\text{C}$ relative to the denaturation level.
It is this capacity for unwinding and subsequent re-pairing of complementary segments that allows researchers to artificially "pair" strands from different sources to evaluate their similarity.
DNA Probe as a Reference Standard
To find a specific sequence in the test sample, a specialized tool called a probe is used.
A probe is a single-stranded nucleic acid molecule. To enable instrumental detection of hybridization, this reference molecule is pre-labeled with special markers—traditionally, radioactive nuclides are used for this purpose.
During the analysis, the probe functions as a strict standard: it interacts only with those segments of the test DNA that possess a high degree of homology (complementarity) to it.
Algorithm of the Classical Reaction
The classical molecular hybridization procedure consists of several sequential steps, each critical for obtaining a reliable result:
- Sample preparation: The microbial DNA under study is subjected to thermal denaturation to separate the double-stranded molecule into individual strands.
- Fixation: One of the resulting strands of the test DNA is firmly anchored onto the surface of a specialized filter.
- Hybridization: The prepared filter with the fixed strand is immersed in a solution containing a radioactively labeled probe.
- Result evaluation: The formation of a hybrid double-stranded helix is considered a positive result. This occurs only if complementarity exists between the microbial DNA and the reference probe.
Microarray Technology
A modern and high-throughput modification of molecular hybridization is microarray technology. The device principle of such a chip is based on using a glass slide (matrix). DNA probes are fixed at strictly defined loci on this slide. A single microarray can contain from 100 to 1,000 types of probes, each specific to a particular microbial taxonomic unit.
Steps of microarray analysis:
- Extraction: Total DNA is extracted from the clinical or laboratory sample.
- Amplification: A stable genetic sequence is amplified. Typically, the 16S rRNA gene serves as the target.
- Labeling: The DNA obtained in the previous step is labeled. Unlike the classical method, fluorochromes or specific enzymes are more commonly used here.
- Hybridization: Direct incubation of the microarray surface with the prepared labeled DNA.
- Washing: Thorough removal of all DNA that has not bound to the probes on the matrix.
- Detection: Determination of the localization of formed hybrids, which appear as glowing spots. Reading is performed using enzyme-linked immunosorbent assay (ELISA) or densitometry. The localization of the luminescence allows for precise identification of the microbial species.