Genomic hybridization is a laboratory method for analyzing an organism’s complete set of DNA, known as the genome. The technique compares an individual’s genetic material against a reference standard, much like comparing two instruction manuals to find where pages have been added, removed, or altered. By highlighting these differences, genomic hybridization helps identify genetic variations that can be associated with certain conditions or traits. This broad overview of the genome can uncover unexpected abnormalities.
The Principle of DNA Hybridization
The foundation of genomic hybridization lies in the unique structure of DNA and the process of hybridization. DNA exists as a double helix, where two long strands are connected by pairs of chemical bases: adenine (A), guanine (G), cytosine (C), and thymine (T). A primary rule of this structure is complementary base pairing, where A always pairs with T, and G always pairs with C.
This structure allows DNA to be manipulated. By applying heat, the bonds holding the two strands together can be broken, causing the double helix to separate, or “denature,” into single strands. When the temperature is lowered, these single strands will naturally seek out and bind to other single strands that have a complementary sequence. This process of reforming a double-stranded molecule is known as hybridization or annealing.
This principle is exploited by mixing single DNA strands from two different sources. They will hybridize where their sequences are similar, and the strength of this new hybrid bond reveals how genetically similar the sources are.
Comparative Genomic Hybridization
Comparative Genomic Hybridization (CGH) provides a panoramic view of the genome to detect differences in the quantity of DNA. This technique analyzes gains or losses of whole chromosomes or large segments of them, making it effective for identifying copy number variations (CNVs). The process avoids the need to culture cells, allowing for a more direct analysis of the sample.
The procedure begins by extracting DNA from two sources: a test sample, such as cells from a tumor, and a normal reference sample. The test DNA is labeled with a green fluorescent molecule and the reference DNA with a red one. These two labeled samples are then mixed in equal amounts and applied to a slide containing a standard set of human chromosomes.
During hybridization, the labeled DNA fragments from both samples compete to bind to their corresponding locations on the reference chromosomes. A fluorescence microscope and computer software then measure the ratio of the two colors. If a region of a chromosome appears yellow, it indicates an equal amount of binding and a normal amount of DNA. An excess of the green signal indicates a gain or amplification, while an excess of red signifies a loss or deletion.
Fluorescence In Situ Hybridization
Fluorescence In Situ Hybridization (FISH) functions more like a targeted searchlight, in contrast to the broad scan of CGH. FISH uses fluorescently tagged DNA fragments called probes, which are designed to be complementary to a specific, known DNA sequence. This allows the probes to bind directly to that location on a chromosome, providing information on its copy number and location.
The process is performed “in situ,” meaning directly on cells or chromosomes fixed onto a glass slide. Both the probe and the target DNA in the cells are denatured with heat to separate them into single strands. When the temperature is lowered, the fluorescent probes hybridize to their specific target sequences. Unbound probes are washed away, and a microscope visualizes where the probes have attached, appearing as bright colored spots.
This specificity makes FISH useful for diagnosing genetic disorders with known chromosomal abnormalities. It can be used to count the number of copies of a particular chromosome to diagnose conditions caused by aneuploidy, such as Down syndrome (Trisomy 21). It is also used to detect structural changes like translocations, where a piece of one chromosome attaches to another, a feature of certain types of leukemia.
Applications in Diagnostics and Research
Genomic hybridization techniques are valuable in both clinical diagnostics and scientific research. In prenatal testing, FISH and CGH are used to screen for chromosomal abnormalities in a fetus. These methods can detect conditions such as Patau syndrome (Trisomy 13), Edward syndrome (Trisomy 18), and Down syndrome (Trisomy 21), providing information for genetic counseling.
In the field of oncology, these methods are used for cancer diagnostics and to guide treatment. CGH can identify the amplification of oncogenes or the deletion of tumor suppressor genes, while FISH can detect specific gene fusions. This information helps develop targeted therapies tailored to the genetic makeup of a patient’s tumor.
Beyond the clinical setting, genomic hybridization is a tool in evolutionary biology. By comparing the genomes of different species, scientists can identify conserved DNA sequences maintained across evolutionary time, as well as unique sequences. This comparative approach provides insights into genetic relationships and the processes of adaptation and speciation.