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Comparative genomic hybridization is a molecular cytogenetic method for analysing copy number variations (CNVs) 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 (a portion of a whole chromosome). This technique was originally developed for the evaluation of the differences between the chromosomal complements of solid tumor and normal tissue,〔Kallioniemi A, Kallioniemi OP, Sudar Da, Rutovitz D, Gray JW, Waldman F, Pinkel D (1992) Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 258:818-821.〕 and has an improved resoIution 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.〔Strachan T, Read AP (2010) Human Molecular Genetics: Garland Science.〕〔Weiss M, Hermsen M, Meijer G, Van Grieken N, Baak J, Kuipers E, Van Diest P (1999) Comparative genomic hybridization. Molecular Pathology 52:243-251.〕 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 a different fluorophores (fluorescent molecules) of different colours (usually red and green), 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 (yellow when the fluorophore labels are red and green) 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 (aCGH) has been developed, allowing for a locus-by-locus measure of CNV with increased resolution as low as 100 kilobases.〔Pinkel D, Albertson DG (2005) Comparative genomic hybridization. Annu Rev Genomics Hum Genet 6:331-354.〕〔de Ravel TJ, Devriendt K, Fryns J-P, Vermeesch JR (2007) What’s new in karyotyping? The move towards array comparative genomic hybridization (CGH). European journal of pediatrics 166:637-643.〕 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 (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 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.〔du Manoir S, Speicher MR, Joos S, Schröck E, Popp S, Döhner H, Kovacs G, Robert-Nicoud M, Lichter P, Cremer T (1993) Detection of complete and partial chromosome gains and losses by comparative genomic in situ hybridization. Human genetics 90:590-610.〕 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〔Kallioniemi OP, Kallioniemi A, Piper J, Isola J, Waldman FM, Gray JW, Pinkel D (1994) Optimizing comparative genomic hybridization for analysis of DNA sequence copy number changes in solid tumors. Genes, Chromosomes and Cancer 10:231-243.〕 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 (DOP-PCR) 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〔Solinas‐Toldo S, Lampel S, Stilgenbauer S, Nickolenko J, Benner A, Döhner H, Cremer T, Lichter P (1997) Matrix‐based comparative genomic hybridization: biochips to screen for genomic imbalances. Genes, Chromosomes and Cancer 20:399-407.〕 and Pinkel et al. in 1998 by use of breast cancer cells.〔Pinkel D, Segraves R, Sudar D, Clark S, Poole I, Kowbel D, Collins C, Kuo W-L, Chen C, Zhai Y (1998) High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays. Nature genetics 20:207-211.〕 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.〔Marquis-Nicholson R, Aftimos S, Hayes I, George A, Love DR (2010) Array comparative genomic hybridization: a new tool in the diagnostic genetic armoury. NZ Med J 123:50-61.〕 Now probes of various origins such as cDNA, genomic PCR products and bacterial artificial chromosomes (BACs) can be used on DNA microarrays which may contain up to 2 million probes.〔 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.〔〔Inazawa J, Inoue J, Imoto I (2004) Comparative genomic hybridization (CGH)‐arrays pave the way for identification of novel cancer‐related genes. Cancer Science 95:559-563.〕 抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』 ■ウィキペディアで「Comparative genomic hybridization」の詳細全文を読む スポンサード リンク
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