Molecular Biology

Molecular Biology Techniques Comparative Genomic Hybridization

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Comparative genomic hybridization is a technique to study changes in copy number of DNA. Classical CGH procedure consists in labeling DNA from the "test" patient" with a particular fluorescent dye that emits green light, while DNA from a normal individual serves as the control and is labeled with a different dye that emits red light when excited by fluorescence. These two DNA samples are mixed in equal amounts and hybridized to chromosome spreads prepared from normal cells arrested in metaphase on a glass slide. After washing off the excess DNA and incubating for adequate time, the fluorescence in different areas of each chromosome is read. The color of a particular region of the chromosome depends upon the ratio of the red to green fluorescence. If the number of copies of a particular DNA sequence is increased in the patient sample compared to the test, then that particular region will appear green, while, conversely, there is a decrease in the number of copies of a particular DNA sequence in the patient DNA sample compared to the test, then, that region of the chromosome will appear red. If there is no change in the number of copies of a particular DNA sequence between the patient and the test, then that region appears yellow. Here we assume that both the test and the patient DNA samples have an equal probability of hybridizing to the DNA on the metaphase chromosomes.
This classical CGH method, though highly effective in being able to detect large areas with copy number change, suffers from certain shortcomings. Perhaps one of the most significant is that it still has a low resolution, usually upto 5-10 M. this means that a change in copy number cannot be detected if this involves a DNA sequence less than 5-10 Mb (5-10 million base pairs) long. These regions can be mapped with other techniques like spectral karyotyping and fluorescent in-situ hybridization, but in case of FISH, prior knowledge of the chromosomal region involved is essential to design a probe specific for it. Another shortcoming of this technique is that it cannot identify balanced translocations, inversions and mosaicism. In other words, this technique is good to identify only changes in number of copies of a particular DNA sequence larger than 5-10 Mb. As is usual, the technique is more sensitive for detecting amplification than deletions. Further, though it can detect changes in number of copies of a particular subregion of the DNA, it cannot detect changes in the number of copies of a particular chromosome (aneuploidy/polyploidy). Other techniques like FISH can help detect these changes.

Microarray Based CGH: a step ahead
Array based CGH is a step ahead of conventional CGH and tries to combine the features of conventional CGH and FISH to achieve greater resolution, does away with the preparation of chromosome spreads and instead involves using cloned DNA fragments spotted onto slides and importantly is highly specific, sensitive and takes less time due to semi-automation of certain steps. Thus it is a high-throughput technique.
In this method, regions of the DNA are chosen which are not repetitive and these are cloned using various systems, most commonly Bacterial artificial chromosomes (BAC). These BAC systems can hold upto 350kb of DNA. These cloned fragments are then spotted onto a glass slide. The sequence of each spot and its location on the chromosome are known. Next, DNA from the patient and a normal control, labeled with green and red dye respectively are hybridized to the array (usually reactions are done in triplicate). After incubation, the fluorescence is read and averaged digitally to obtain the average relative fluorescence ratio between the patient and normal samples. This ratio is usually expressed as log values (for e.g. 0.5 times logarithmic change corresponds to a 2-fold change (increase/decrease)). A value more than 2-fold is considered significant. By cloning overlapping regions of DNA, we can narrow down the region which is differentially amplified/deleted in the study subject. By using this technique, it has been possible to identify change in copy number of a region as small as 40 kb (by cloning smaller and smaller fragments of DNA).

Advantages of array CGH
The array CGH, like conventional CGH allows genomewide screening for changes in copy number. However, unlike the conventional CGH, the array method has a higher resolution, thus it is possible to pinpoint exactly the region where the change has occurred.

Some applications of CGH
The aberrations that can be detected by CGH can be divided for the sake of convenience into numerical and structural types.
Numerical aberrations include the Down's syndrome which is characterized classically by a trisomy of the 20th chromosome, or in other cases by duplication of only a part of the chromosome, unbalanced rearrangements of the chromosome or mosaicism of the chromosome. This can be detected by CGH even in cases where FISH is negative.
Structural aberrations such as Cri du chat syndrome are characterized by changes in the copy number of a small region of a chromosome 5p (short arm). Array CGH is very useful to look at submicroscopic levels in order to pinpoint the copy number change (microdeletion usually) which could previously be achieved only by FISH. For instance, a structural change in chromosome 15q11-13 when inherited from the father results in Prader Willi syndrome, while a similar anomaly inherited from the mother results in Angelman syndrome. Array CGH could correctly map the microdeletion in the region. Array CGH can also be used to map with accuracy the critical region of a chromosome involved in a particular genetic disease. For instance, it was known that a deletion involving 18q was responsible for the congenital aural atresia syndrome, but scientists could not narrow down the region critically involved in this condition. Using array CGH, a 5mb region on chromosome 18q22.3-23 as mapped in children suffering from this rare anomaly and seems to be the sought after region. This technique has also been used to study the relationship between a particular genotype and the phenotype associated with it. For instance, Neurofibromatosis type 2 disease has varying grades of severity which were reported to be correlated with different mutations in chromosome 22. However, a CGH array based study showed that there was no relationship between the various deletion anomalies of chromosome 22 and the severity of this disease, although the size of the deleted region varied from 40kb to 6.6 Mb.

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