Comparative genomic hybridization
Comparative genomic hybridization is a molecular cytogenetic method for analysing copy number variations relative to ploidy level in the DNA of a test sample compared to a reference sample, without the need for culturing cells. The aim of this technique is to quickly and efficiently compare two genomic DNA samples arising from two sources, which are most often closely related, because it is suspected that they contain differences in terms of either gains or losses of either whole chromosomes or subchromosomal regions. This technique was originally developed for the evaluation of the differences between the chromosomal complements of solid tumor and normal tissue, 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 which are limited by the resolution of the microscope utilized.
This is achieved through the use of competitive fluorescence in situ hybridization. In short, this involves the isolation of DNA from the two sources to be compared, most commonly a test and reference source, independent labelling of each DNA sample with fluorophores of different colours, denaturation of the DNA so that it is single stranded, and the hybridization of the two resultant samples in a 1:1 ratio to a normal metaphase spread of chromosomes, to which the labelled DNA samples will bind at their locus of origin. Using a fluorescence microscope and computer software, the differentially coloured fluorescent signals are then compared along the length of each chromosome for identification of chromosomal differences between the two sources. A higher intensity of the test sample colour in a specific region of a chromosome indicates the gain of material of that region in the corresponding source sample, while a higher intensity of the reference sample colour indicates the loss of material in the test sample in that specific region. A neutral colour indicates no difference between the two samples in that location.
CGH is only able to detect unbalanced chromosomal abnormalities. This is because balanced chromosomal abnormalities such as reciprocal translocations, inversions or ring chromosomes do not affect copy number, which is what is detected by CGH technologies. CGH does, however, allow for the exploration of all 46 human chromosomes in single test and the discovery of deletions and duplications, even on the microscopic scale which may lead to the identification of candidate genes to be further explored by other cytological techniques.
Through the use of DNA microarrays in conjunction with CGH techniques, the more specific form of array CGH has been developed, allowing for a locus-by-locus measure of CNV with increased resolution as low as 100 kilobases. This improved technique allows for the aetiology of known and unknown conditions to be discovered.
History
The motivation underlying the development of CGH stemmed from the fact that the available forms of cytogenetic analysis at the time 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 first report of CGH analysis was by Kallioniemi and colleagues in 1992 at the University of California, San Francisco, who utilised CGH in the analysis of solid tumors. They achieved this by the direct application of the technique to both breast cancer cell lines and primary bladder tumors in order to establish complete copy number karyotypes for the cells. They were able to identify 16 different regions of amplification, many of which were novel discoveries.
Soon after in 1993, du Manoir et al. reported virtually the same methodology. 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. It was concluded that the fluorescence ratios obtained were accurate and that differences between genomic DNA from different cell types were detectable, and therefore that CGH was a highly useful cytogenetic analysis tool.
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. However, in 1994 a review was published which described an easily understood protocol in detail and the image analysis software was made available commercially, which allowed CGH to be utilised all around the world.
As new techniques such as microdissection and degenerate oligonucleotide primed polymerase chain reaction became available for the generation of DNA products, it was possible to apply the concept of CGH to smaller chromosomal abnormalities, and thus the resolution of CGH was improved.
The implementation of array CGH, whereby DNA microarrays are used instead of the traditional metaphase chromosome preparation, was pioneered by Solinas-Tolodo et al. in 1997 using tumor cells and Pinkel et al. in 1998 by use of breast cancer cells. This was made possible by the Human Genome Project which generated a library of cloned DNA fragments with known locations throughout the human genome, with these fragments being used as probes on the DNA microarray. Now probes of various origins such as cDNA, genomic PCR products and bacterial artificial chromosomes can be used on DNA microarrays which may contain up to 2 million probes. Array CGH is automated, allows greater resolution 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.
Image:CGH schema.jpg|right|thumb|Figure 1. Schematic of CGH protocol
Basic methods
Metaphase slide preparation
The DNA on the slide is a reference sample, and is thus obtained from a karyotypically normal man or woman, though it is preferential to use female DNA as they possess two X chromosomes which contain far more genetic information than the male Y chromosome. Phytohaemagglutinin stimulated peripheral blood lymphocytes are used. 1mL of heparinised blood is added to 10ml of culture medium and incubated for 72 hours at 37 °C in an atmosphere of 5% CO2. Colchicine is added to arrest the cells in mitosis, the cells are then harvested and treated with hypotonic potassium chloride and fixed in 3:1 methanol/acetic acid.One drop of the cell suspension should then be dropped onto an ethanol cleaned slide from a distance of about 30 cm, optimally this should be carried out at room temperature at humidity levels of 60–70%. 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. Different donors should be tested as hybridization may be variable. Commercially available slides may be used, but should always be tested first.
Isolation of DNA from test tissue and reference tissue
Standard phenol extraction is used to obtain DNA from test or reference tissue, which involves the combination of Tris-Ethylenediaminetetraacetic acid and phenol with aqueous DNA in equal amounts. 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.May be completed using DNA isolation kits available commercially which are based on affinity columns.
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. 0.5-1 μg of DNA is sufficient for the CGH experiment, though if the desired amount is not obtained DOP-PCR may be applied to amplify the DNA, however it in this case it is important to apply DOP-PCR to both the test and reference DNA samples to improve reliability.
DNA labelling
is used to label the DNA and involves cutting DNA and substituting nucleotides labelled with fluorophores or biotin or oxigenin to have fluophore conjugated antibodies added later. 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.Blocking
Unlabelled Life Technologies Corporation's Cot-1 DNA is added to block normal repetitive DNA sequences, particularly at centromeres and telomeres, as these sequences, if detected, may reduce the fluorescence ratio and cause gains or losses to escape detection.Hybridization
8–12μl of each of labelled test and labelled reference DNA are mixed and 40 μg Cot-1 DNA is added, then precipitated and subsequently dissolved in 6μl of hybridization mix, which contains 50% formamide to decrease DNA melting temperature and 10% dextran sulphate to increase the effective probe concentration in a saline sodium citrate solution at a pH of 7.0.Denaturation of the slide and probes are carried out separately. The slide is submerged in 70% formamide/2xSSC for 5–10 minutes at 72 °C, while the probes are denatured by immersion in a water bath of 80 °C for 10 minutes and are immediately added to the metaphase slide preparation. This reaction is then covered with a coverslip and left for two to four days in a humid chamber at 40 °C.
The coverslip is then removed and 5 minute washes are applied, three using 2xSSC at room temperature, one at 45 °C with 0.1xSSC and one using TNT at room temperature. The reaction is then preincubated for 10 minutes then followed by a 60-minute, 37 °C incubation, three more 5 minute washes with TNT then one with 2xSSC at room temperature. The slide is then dried using an ethanol series of 70%/96%/100% before counterstaining with DAPI, for chromosome identification, and sealing with a coverslip.