Comparative genomic hybridization
This technique was originally developed for the evaluation of the differences between the chromosomal complements of solid tumor and normal tissue,[1] and has an improved resolution of 5–10 megabases compared to the more traditional cytogenetic analysis techniques of giemsa banding and fluorescence in situ hybridization (FISH) which are limited by the resolution of the microscope utilized.
The motivation underlying the development of CGH stemmed from the fact that the available forms of cytogenetic analysis at the time (giemsa banding and FISH) were limited in their potential resolution by the microscopes necessary for interpretation of the results they provided.
Furthermore, giemsa banding interpretation has the potential to be ambiguous and therefore has lowered reliability, and both techniques require high labour inputs which limits the loci which may be examined.
The authors painted a series of individual human chromosomes from a DNA library with two different fluorophores in different proportions to test the technique, and also applied CGH to genomic DNA from patients affected with either Downs syndrome or T-cell prolymphocytic leukemia as well as cells of a renal papillary carcinoma cell line.
[6] Initially, the widespread use of CGH technology was difficult, as protocols were not uniform and therefore inconsistencies arose, especially due to uncertainties in the interpretation of data.
[3] However, in 1994 a review was published which described an easily understood protocol in detail[7] and the image analysis software was made available commercially, which allowed CGH to be utilised all around the world.
[10] Array CGH is automated, allows greater resolution (down to 100 kb) than traditional CGH as the probes are far smaller than metaphase preparations, requires smaller amounts of DNA, can be targeted to specific chromosomal regions if required and is ordered and therefore faster to analyse, making it far more adaptable to diagnostic uses.
Slides should be evaluated by visualisation using a phase contrast microscope, minimal cytoplasm should be observed and chromosomes should not be overlapping and be 400–550 bands long with no separated chromatids and finally should appear dark rather than shiny.
Slides then need to be air dried overnight at room temperature, and any further storage should be in groups of four at −20 °C with either silica beads or nitrogen present to maintain dryness.
This is followed by separation by agitation and centrifugation, after which the aqueous layer is removed and further treated using ether and finally ethanol precipitation is used to concentrate the DNA.
[3] Preferentially, DNA should be extracted from fresh or frozen tissue as this will be of the highest quality, though it is now possible to use archival material which is formalin fixed or paraffin wax embedded, provided the appropriate procedures are followed.
It is then important to check fragment lengths of both test and reference DNA by gel electrophoresis, as they should be within the range of 500kb-1500kb for optimum hybridization.
The microscope must provide uniform illumination without chromatic variation, be appropriately aligned and have a "plan" type of objective which is apochromatic and give a magnification of x63 or x100.
[14] Array CGH has proven to be a specific, sensitive, fast and high-throughput technique, with considerable advantages compared to other methods used for the analysis of DNA copy number changes making it more amenable to diagnostic applications.
[15] As of 2006[update], even high-resolution CGH (HR-CGH) arrays are accurate to detect structural variations (SV) at resolution of 200 bp.
In array CGH, these targets can be genomic fragments cloned in a variety of vectors (such as BACs or plasmids), cDNAs, or oligonucleotides.
The use of BACs provides sufficient intense signals to detect single-copy changes and to locate aberration boundaries accurately.
These arrays currently yield a high spatial resolution, but the number of cDNAs is limited by the genes that are encoded on the chromosomes, and their sensitivity is low due to cross-hybridization.
[17] Conventional CGH has been used mainly for the identification of chromosomal regions that are recurrently lost or gained in tumors, as well as for the diagnosis and prognosis of cancer.
For example, gains of chromosomal regions lq, 3q and 8q, as well as losses of 8p, 13q, 16q and 17p, are common to a number of tumor types, such as breast, ovarian, prostate, renal and bladder cancer (Figure.
Cri du Chat (CdC) is a syndrome caused by a partial deletion of the short arm of chromosome 5.
These results demonstrate that conventional CGH is a reliable technique in detecting structural aberrations and, in specific cases, may be more efficient in diagnosing complex abnormalities.
However, array CGH is also suitable for the analysis of DNA copy number aberrations that cause human genetic disorders.
In a recent study, array CGH has been implemented to identify regions of chromosomal aberration (copy-number variation) in several mouse models of breast cancer, leading to identification of cooperating genes during myc-induced oncogenesis.
As a proof of principle Vissers et al. (2003) constructed a genome wide array with a 1 Mb resolution to screen three patients with known, FISH-confirmed microdeletion syndromes, including one with PWS.
Though not yet a widely employed technique, the use of array CGH as a tool for preimplantation genetic screening is becoming an increasingly popular concept.
This makes array CGH a promising tool to reduce the incidence of life altering conditions and improve success rates of IVF attempts.
[30] In addition, chromosomal regions with short repetitive DNA sequences are highly variable between individuals and can interfere with CGH analysis.
Although CGH has proven to be a useful and reliable technique in the research and diagnostics of both cancer and human genetic disorders, the applications involve only gross abnormalities.
The standard resolution varies between 1 and 5 Mb, but can be increased up to approximately 40 kb by supplementing the array with extra clones.
