Understanding the FISH Technique Sequence

The question asks for the correct order of steps involved in the Fluorescence In Situ Hybridization (FISH) technique. FISH is a powerful molecular cytogenetic technique used to detect and locate specific DNA sequences within a cell or tissue sample. It utilizes fluorescently labeled probes that bind specifically to target DNA sequences.

FISH Technique Steps: Correct Order Explained

The correct sequence for performing the FISH technique involves preparing the sample, making the DNA accessible, allowing the probe to bind, and finally detecting the result. Based on this, the correct order is C, A, B, D.

• C. Fixation of cells on a slide: This is the crucial first step. Cells or tissues are treated to preserve their structure and attach them firmly to a microscope slide. This preparation ensures the cells remain intact throughout the subsequent experimental procedures.
• A. Denaturation of DNA: Before a probe can bind to its target sequence, the double-stranded DNA within the cells (and often the probe itself) needs to be separated into single strands. This process, called denaturation, is typically achieved by heating the slide or using chemical agents. It makes the DNA bases accessible for the probe.
• B. Hybridization of probe to target DNA: Once the DNA is denatured (single-stranded), the fluorescently labeled probe, which is designed to be complementary to the specific target DNA sequence, is applied to the slide. The probe then searches for and binds (hybridizes) to its matching sequence on the target DNA under specific temperature and incubation conditions.
• D. Detection of signal using a fluorescence microscope: After hybridization, any unbound probes are washed away. The slide is then examined using a fluorescence microscope. The microscope excites the fluorophore attached to the bound probe, causing it to emit light of a specific color. This fluorescence signal visually indicates the location and presence of the target DNA sequence within the cells.

Therefore, the sequence C -> A -> B -> D represents the standard workflow for conducting a FISH experiment.