Hybridization
Hybridization is the re-annealing phase where the “search engine” meets the “database.” Once the DNA of the probe and the specimen have been separated into single strands (denaturation), the temperature is lowered to allow them to pair up. The goal is for the fluorescently labeled probe DNA to find and bind specifically to its complementary sequence on the patient’s chromosome, forming a stable DNA:DNA hybrid. This process is driven by the principles of molecular thermodynamics
The Thermodynamics of Hybridization
Hybridization is a competition between Probe-Target binding (what we want) and Target-Target re-annealing (the original DNA zipping back together). To favor the probe, two conditions are met:
- Probe Excess: A massive molar excess of probe is added to the slide to saturate the target sites
- Cot-1 DNA (Blocking Repetitive Sequences): Most probes are generated from genomic DNA, which naturally contains highly repetitive sequences (Alu repeats, LINES, SINES) found everywhere in the genome. If not blocked, the probe would bind randomly across all chromosomes, lighting up the whole nucleus green. Cot-1 DNA is unlabeled repetitive DNA added to the probe mix. It binds to the repetitive sequences on the probe first, effectively “masking” them so that only the unique gene-specific sequence is free to hybridize to the chromosome
Temperature & Time
-
Temperature (\(37^\circ\text{C}\))
- Hybridization typically occurs at \(37^\circ\text{C}\). This is the optimal temperature for specific annealing in the presence of Formamide
- Too High: The thermal energy breaks the hydrogen bonds as soon as they form. No signal
- Too Low: Non-specific binding occurs. The probe binds to sequences that are “mostly” similar but not perfect matches (mismatching). This creates high background noise (cross-hybridization)
-
Time (Incubation)
- Standard: Overnight (14–16 hours). This allows sufficient time for the probe molecules to diffuse through the cell/tissue and find their target
- Rapid Hybridization: Newer “Fast FISH” buffers use chemical accelerants (e.g., ethylene carbonate) to reduce this time to 1–2 hours, enabling same-day STAT results (e.g., for newborn aneuploidy or APL leukemia)
The Humidity Chamber
During the long incubation at \(37^\circ\text{C}\), the liquid hybridization buffer (which is only 10 \(\mu\)L) is at risk of evaporating. If the slide dries out, the probe concentrates, precipitates, and binds irreversibly to the glass, causing a massive fluorescent “mess” that ruins the slide
- Sealing: The coverslip is sealed with rubber cement to create a closed chamber
- Environment: The slides are placed in a Humidified Chamber (a dark box with water-soaked paper towels) inside the incubator to maintain 100% relative humidity, preventing evaporation
Stringency (The Wash)
Strictly speaking, hybridization continues into the Post-Hybridization Wash. After incubation, the slide is covered in excess probe that is loosely bound to non-target DNA. The wash step removes this non-specific binding
- Stringency: This refers to the strictness of the wash conditions. High stringency removes everything except perfect matches
-
Factors
- High Temperature (\(72^\circ\text{C}\)): High stringency (removes weak bonds)
- Low Salt Concentration (0.4x SSC): High stringency (salt stabilizes DNA; low salt destabilizes weak bonds)
- Balance: If the wash is too stringent (too hot/low salt), the true signal is washed away. If too lenient (too cold/high salt), the background noise remains high