Study of Chromosomes


G-banding was mentioned above as a method to match up homologous chromosomes. When the Giemsa stain is applied to the chromosomes the homologous chromosome pairs will have the same banding pattern as one another. Once the chromosomes are paired, a trained professional can look at them under a microscope and detect any large abnormalities.

Fluorescent in situ Hybridization (FISH)

Sometimes a genetic change is too small to visualize through G-banding alone. For example a disease such as 22q11.2 Deletion Syndrome which is caused by a deletion on chromosome 22 might not be detected by G-banding. Other methods have been created to take advantage of new technology and equipment and recognize these smaller abnormalities.

One method is Fluorescent In Situ Hybridization (FISH). The FISH technique uses probes (strands of DNA sequence) that are complimentary to and bind to specific sequences on the person’s chromosomes. Each probe is labeled with a fluorescent dye.

To test a patient’s DNA it first needs to be separated into single strands through a process called denaturation. Then the chosen probe(s) will be added to the patient’s DNA and allowed to hybridize or bind. Next the DNA sample is allowed to re-nature into a double helix (with the fluorescent probes bound).

When the sample is visualized under the proper light the areas where the probes are bound will look like bright colored dots. The number of colored dots indicates the number of copies the patient has of that specific DNA sequence. Typically the normal number of copies would be two. If a patient had two fluorescent dots, there would not be a mutation and would be two normal copies of that region of DNA. One dot would mean that there is a mutation on one of the two copies and the patient is heterozygous for that mutation (one changed copy and one unchanged copy). No fluorescent dots would mean that both copies of that area of DNA had mutations and the patient is homozygous for that mutation (two changed copies and no unchanged copies).

For example, FISH is commonly used to detect the deletion causing 22q11.2 Deletion Syndrome. A probe that is complementary to the normal 22q11.2 sequence is allowed to bind with the patient’s DNA. When the FISH result is visualized a patient with the deletion will have only one fluorescent colored dot while a person without the deletion would have two.

There are two different types of FISH, each named for the phase in the cell cycle the cells are in at the time of the test. Metaphase FISH looks at cells that are currently dividing and in the metaphase stage of the cell cycle. Chromosomes in the metaphase stage are tightly coiled and look like the chromosomes in the karyotype pictures above.  

The other type of FISH is Interphase FISH. The cells analyzed through this process do not have to be actively dividing and are in the interphase of the cell cycle. The same process is used for interphase and metaphase FISH techniques, but the resulting image is very different.  Because chromosomes are not condensed during interphase, interphase FISH does not allow the cytogeneticist to see the actual chromosome structures. All that can be seen are dots of fluorescence on a dark background (pictured below). However, the dots have the same meaning as with metaphase FISH. One important benefit of interphase FISH is that it can be done more quickly since the cells do not have to be in any specific stage of the cell cycle. Interphase FISH is commonly used to test amniotic fluid cells (taken from the area around an unborn baby) for chromosome abnormalities. The cell’s chromosomes are denatured and hybridized with DNA probes for the chromosomes 13, 18, 21, X, and Y. The results of this screen will tell the doctor how many of each of these chromosomes the baby has by counting the different colored dots that appear. The chromosomes looked at in this screening test are those that cause the most common chromosome abnormalities. See below for a discussion on prenatal testing.

FISH Image 1 FISH Image 2
Above: Images of interphase FISH. Both are samples from the same patient. Colored probes on the left indicate two of chromosome 21 (red) and three of chromosome 13 (green). Colored probes on the right indicate two of chromosome 18 (aqua), one chromosome X (green) and one chromosome Y (red). This patient is a male with trisomy 13.

Images courtesy of GeneCare Medical Genetics Center

Array Comparative Genomic Hybridization (aCGH)

Another way to analyze genetic information is a technique called Array Comparative Genomic Hybridization (ArrayCGH). ArrayCGH uses microarray technology to look at genetic information in more detail than can be done with G-Banding or FISH. ArrayCGH looks at many specific chromosome locations to detect areas of extra or missing information (gains and losses). A gain or loss in a person’s DNA sequence is called a copy number variation.

A patients DNA sample is allowed to bind to a glass slide containing numerous probes (complimentary segments of DNA). The patient’s DNA is fluorescently labeled as well as a reference DNA sample, used as a normal control. Different fluorescent colors are used for the patient sample and the reference DNA. For example, the patient sample DNA could be colored red and the control DNA colored green. After the samples have bound the machine calculates the fluorescent intensity ratio for each point on the slide. Differences between the two sets of DNA can be detected and abnormal regions of the patient’s genome can be identified. A area on the slide where more patient DNA binds than control DNA will fluoresce red, whereas a spot that binds more control DNA than sample DNA will fluoresce green. A spot with an equal number of both will fluoresce yellow because it has an equal amount of red and green.

The results of the experiment are translated onto a graph, shown below. A normal arrayCGH reading will look like a straight line. This is because the patient’s DNA did not have any gains or losses to make it bind differently than the reference DNA. However, an abnormal aCGH graph reading will not be a straight line, but will have peaks, because the two samples did not bind equally to all of the probes.

CGH Image

Above: Sample array CGH plots for chromosome number 22. The graph on the left is from a patient with normal chromosomes 22. The graph on the right is from a patient with DiGeorge Syndrome. This syndrome is caused by a deletion on chromosome 22, illustrated by the peak on the graph.

Image courtesy of University of Alabama Birmingham, Department of Genetics

The detection of these gains and losses in a person’s genome is important because scientists are currently learning more and more ways that these copy number variantions (CNV) and genetic changes relate to human disease. A recent HudsonAlpha Biotech 101 article discusses what a copy number variant is and its implications on human genetics and disease. You can find the article here.

Microarrays can also be used to determine which genes are expressed in a particular cell. When a gene is expressed, the cell transcribes the gene into mRNA which is used to make protein. The mRNA found in a cell is like a roadmap of what genes are being expressed. mRNA can be extracted from the cell and reverse transcribed back into DNA which can then be tested on a microarray to identify the genes. Below is an interactive online activity about microarray analysis of cancer cells.

Microarray Activity Image Left: Link to a website about using karyotypes to predict genetic disorders. There are click-through animations for events such as abnormal meiosis and an interactive quiz about genetic disorders and their karyotypes.

“DNA Microarray Virtual Lab.” Genetic Science Learning Center. 2008. University of Utah. 10 December 2008.

Pre-natal Testing

To perform tests such as FISH or aCGH, the person’s DNA must be extracted from a sample of cells. Many times this is a blood sample collected through a simple blood draw. As mentioned above, sometimes genetic analysis is performed on a pregnancy and in this situation a blood sample is not accessible.  However, alternative procedures for collecting fetal cells have been developed and include amniocentesis and chorionic villus sampling (CVS).

Amniocentesis is a process where a needle, guided by ultrasound, is inserted through the mother’s stomach into the amniotic sac and withdraws a small amount of amniotic fluid from around the baby. This fluid contains some fetal skin cells that have been sloughed off throughout the pregnancy. DNA can be extracted from these cells in a laboratory and be analyzed for chromosomal and genetic abnormalities. Amniocentesis is generally performed starting at 14 weeks gestation. An explanation and illustration of the procedure can be found at:

Chorionic villus sampling (CVS) is a similar procedure to amniocentesis but can be performed earlier during pregnancy, between 10 and 13 weeks. A thin catheter is inserted either through the mother’s stomach or through her cervix depending on which location is safer for the baby. In this procedure, a sample is taken from the chorionic villi which are finger-like growths in the placenta. These cells contain the same genetic information as the fetus. DNA is extracted from the chorionic villi cells and can be analyzed for chromosomal and genetic abnormalities. Illustrations of chorionic villi and both methods of the procedure can be found at:

Both of these procedures are invasive and carry a small risk of miscarriage. These tests also cannot detect every genetic disorder but are accurate in detecting chromosomal abnormalities such as Down Syndrome, and large chromosomal rearrangements, by looking at the baby’s karyotype. Also, if a specific genetic mutation is known to be in a family, a baby’s DNA can be tested specifically for this mutation. Cystic Fibrosis and Tay-Sachs disease are examples of disorders that would not be picked up by looking at a karyotype, but could be detected by specifically looking at the genes involved if the parents are known to be carriers.

There are many reasons parents might decide to have prenatal genetic testing. For example, there may be a history of a specific genetic condition in the family, or there may be an increased risk for a chromosome abnormality based on the mother’s age, ultrasound findings, or results from a screening test.