Summary of Fluorescence in situ hybridization
Transcript for Fluorescence in situ hybridization
Content Reviewers:Jessica Reynolds, MS, Evan Debevec-McKenney, Victoria Silvia Recalde, Viviana Popa, MD
Fluorescence in situ hybridization
Fluorescence in situ hybridization is a cytogenetic technique that uses fluorescent DNA segments, called “probes”, to bind to a known DNA sequence.
It’s used to localize particular DNA sequences, or lack thereof, on a chromosome in order to detect chromosomal abnormalities, or mutations; like deletion, duplication, or translocation of a DNA segment; which may be the underlying cause of a genetic disease.
Ok, now, our DNA is like a library, found in the nucleus of our cells, that carries our genetic information.
On the molecular level, DNA is made up of two strands of nucleotides that are coiled around one another to form a double helix.
There are four different nucleotides: adenine, or A, thymine, or T, cytosine, or C and guanine, or G. A binds with T, and C binds with G; nucleotides on opposite strands form hydrogen bonds to keep the two strands together.
To fit inside the nucleus, DNA wraps around proteins which further condense to form chromatin fibers.
These chromatin fibers are loosely or tightly packed depending on the phase of the cell’s cycle.
The cell cycle represents a series of events that somatic cells, that is, all cells besides the gametes, go through from the moment they’re formed until the moment they divide into two identical daughter cells.
During early interphase, chromatin fibers float in a loose state inside the nucleus, like DNA-rich noodles.
Each of the chromatin noodles represents a single DNA molecule.
Now, during later interphase, when the cell prepares for mitosis, or cellular division, the DNA noodles replicate and chromatin condenses to form chromosomes.
Remember that human somatic cells have 23 pairs of chromosomes, so 46 chromosomes in total.
And right before mitosis, each chromosome carries two identical DNA molecules, called chromatids.
Chromatids join together in the center in a region called the centromere, which in turn divides both chromatids into a short “p” arm, and a long “q” arm.
Finally, during mitosis, the chromosomes condense, so they can be observed in more detail - and they’re at their most condensed during a phase of mitosis called metaphase, when they neatly align in the middle of the cell, like 46 little X shapes - where each side of the X represents a chromatid.
Having said that, let’s say, for example, that you suspect an individual has cri-du-chat, or 5p- syndrome, which is when one chromosome 5 is missing the tip of its short arm.
A fluorescence in situ hybridization, or FISH done on metaphase chromosomes can show if that part is missing, and confirm the diagnosis.
With metaphase FISH, first, a double-stranded DNA segment is prepared in the lab, which is made up of the same nucleotide sequence as the tip of chromosome 5’s short arm.
This can be done thanks to the human genome project, which determined the nucleotide sequence of the entire human’s DNA… great time to be in science, folks!
Okay, so this DNA segment is called a probe, and it’s actually made up of fluorescent nucleotides, which are labeled with colored molecules called fluorophores.
Not only is that super cool, but it also makes fluorescent nucleotides look like colored spots under fluorescent microscopy.
Fluorophores come in different colors, so you can take a pick…
In this case, let’s choose red.
Next, a sample of the person’s cells are put on a glass slide.
They contain the chromosomes with the target DNA.
Now, both the target DNA and the probes are heated to about 95ºC.
This step is called denaturation, because the heat breaks the hydrogen bonds that normally keep DNA strands together.
Now the separated strands can be hybridized, which means that each DNA-strand of the probe binds to its complementary DNA sequence on the chromosome.
So, now the chromosome has been tagged with a fluorescent “red flag”.
So, if everything goes as is expected and both of a person’s chromosome 5s have their entire short arms, there would be four red spots: two spots for each chromosome, one for each chromatid.