Molecular hybridization method

Physicochemical basis of the method

The foundation of molecular hybridization is a unique physicochemical property of the DNA molecule. It is capable of reversible structural changes in response to environmental temperature fluctuations.

Key processes occurring with DNA:

• Denaturation. This is the process of strand unwinding. It is triggered by heating the medium to $90^\circ\text{C}$, and the reaction must take place under alkaline conditions. As a result, the double-stranded structure separates into individual strands.
• Renaturation. This is the process of restoring the original double-stranded helix. For the strands to reconnect, the temperature must be lowered by $10^\circ\text{C}$ from the denaturation level.

It is this ability to unwind and subsequently re-anneal complementary regions that allows researchers to artificially "pair" strands from different sources to assess their similarity.

DNA probe as a reference standard

To find a specific sequence in the test sample, a special tool is used — a probe.

The 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 regions of the test DNA that exhibit a high degree of homology (complementarity) to it.

Classic reaction workflow

The classical molecular hybridization procedure consists of several sequential steps, each of which is critical for obtaining a reliable result:

1. Sample preparation. The test microbial DNA is subjected to thermal denaturation to unwind the double-stranded molecule into individual strands.
2. Fixation. One of the resulting test DNA strands is firmly immobilized on the surface of a special filter.
3. Hybridization. The prepared filter with the immobilized strand is immersed in a solution containing a radioactively labeled probe.
4. Result evaluation. A positive result is indicated by the formation of a hybrid double-stranded helix. This occurs only if there is complementarity between the microbial DNA and the reference probe.

Microarray technology

A modern and high-throughput modification of molecular hybridization is microarray technology. The design principle of such a chip is based on the use of a glass plate (matrix). DNA probes are fixed at strictly defined loci on this plate. A single microarray can contain from 100 to 1,000 types of probes, each specific to a particular microbial taxonomic unit.

Stages of microarray analysis:

1. Extraction. Total DNA is extracted from the provided clinical or laboratory sample.
2. Amplification. A stable genetic sequence is amplified. Typically, the 16S rRNA gene serves as the target.
3. Labeling. The DNA obtained in the previous step is labeled. Unlike the classical method, fluorochromes or specific enzymes are more commonly used here.
4. Hybridization. Direct treatment of the microarray surface with the prepared labeled DNA.
5. Washing. Thorough removal of all DNA that has not bound to the probes on the matrix.
6. Detection. Determination of the localization of the formed hybrids. They appear as glowing spots. Reading is performed using enzyme-linked immunosorbent assay (ELISA) or densitometry. The localization of the signal allows for precise identification of the microbial species.

Briefly: answers to topical questions

Class and exam questions

What else to study on this topic

• Mechanisms of denaturation and renaturation of nucleic acids
• Principles of radioisotope labeling of DNA probes
• The role of the 16S rRNA gene in bacterial taxonomy and identification
• Use of densitometry and ELISA for microarray result detection

Other topics in the "Microbial Genetics" section

Prepared by WhiteCoat Pro faculty in accordance with the medical school curriculum. For educational purposes only.