Fluorescence in situ hybridization
Definitions & Key takeaways
Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes to detect and localize the presence of specific DNA sequences on chromosomes. This technique can be used to detect chromosomal abnormalities, such as translocations, duplications, insertions, and deletions. FISH can also be used to identify and map the location of gene sequences on chromosomes.
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.
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.
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.
But since chromatids are really close together, they’re difficult to differentiate, so it shows up as a total of two red spots: only one spot for each chromosome 5.
Now, if microscopy only shows one fluorescent red spot, two things are possible. One is that an entire chromosome of the homologous pair is missing, and the other is that only part of the p arm is missing.
This can be easily solved by adding, from the beginning, a second “green” probe that binds to the tip of the q arm of the same chromosome.
So, if there’s two green spots, but only one red spot; that means there’s one chromosome 5 that is complete, and one that only lacks part of the p arm.
This is called a deletion, it confirms the diagnosis of Cri du Chat, or 5p- syndrome.Now, let’s take a break from all this genetic stuff and take up a little knitting class.
Let’s say you take 10cm of white wool and stain about 1cm with a red colorant. It is easy to see the dyed segment right?
But if you roll the wool in a knitting needle, that 1 cm will look smaller and harder to see. The same goes for chromatin and chromosomes.
It is easier to observe a stained segment of DNA that is loosely packed, like the chromatin during interphase, than the same segment tightly packed into chromosomes during metaphase.
So if you want to detect chromosomal abnormalities that involve a relatively smaller DNA segment, such as duplications or translocations, an interphase FISH can get you a better picture.
The process is basically the same: get the probe, the person’s sample, denature both, and then hybridize them to make the DNA segments on the chromosomes visible.
But the difference is how they look under the microscope: with metaphase FISH, chromosomes are condensed (think pre-cooked ramen), whereas with interphase FISH, they look more like chromatin noodles.Now, Charcot-Marie-Tooth disease is an example of duplication - specifically, duplication of a DNA segment on one of the chromosomes 17.
For example, the chromosome that came from mom has it’s normal segment, and the chromosome that came from dad has two copies of the same segment; making a total of three fluorescent “red” spots under the microscope.
And just like with metaphase FISH, we need to see if there’s really just two chromosomes 17 and three segments, or if there are actually three chromosomes 17.
So a second “green” probe can be added from the beginning. This probe targets a distant segment of DNA on the same chromosome pair.
So if there’s only two green spots, that means only two chromosomes; one of them carrying a duplication.Finally, a classic example of translocation is the Philadelphia chromosome which is a chromosome 22 with a bit of chromosome 9 on it.
Right where they come together, there’s a fusion between the BCR gene, initially in chromosome 22; and the ABL gene, originally in chromosome 9.
So there’s a fusion gene BCR-ABL which leads to uncontrolled cell division that results in a type of leukemia. Here, two different probes are used again: a red one that binds to the DNA segment with the ABL gene on chromosome 9, and a green one that binds to the segment with the BCR gene on chromosome 22.
So, under the microscope, there’s a red spot, that corresponds to the DNA segment normally located on one chromosome 9; a green spot, that corresponds to the DNA segment normally located on one chromosome 22; and finally, there’s a red and green spot on the other chromosome 22 with a bit of chromosome 9 - or the Philadelphia chromosome.##SummaryAll right, as a quick recap… Fluorescence in situ hybridization is a cytogenetic technique that uses fluorescent probes to localize a DNA segment on a chromosome.
The basic steps are: to get the fluorescent probe, then the person’s sample, denature both, and then hybridize them to make the DNA segments on the chromosomes or chromatin visible.
Metaphase FISH is used to analyze chromosomes and detect chromosomal abnormalities that affect a relatively large DNA segment, while interphase FISH is used to analyze chromatin and detect chromosomal abnormalities that affect a relatively small DNA segment.
The mutations that can be detected are: deletions, like in Cri du Chat syndrome; duplications, like in Charcot-Marie-Tooth disease; and translocations, like in Philadelphia chromosome.
a relatively small DNA segment The mutations that can be detected are deletions like in crich syndrome duplications like in Charcot Marie tooth disease and transcations
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