cytogenetic technique for the detection of chromosomal copy number changes on a genome wide and high-resolution scale. array cgh compares the patient's genome against a reference genome and identifies differences between the two genomes, and hence locates regions of genomic imbalances in the patient, utilizing the same principles of competitive fluorescence in situ hybridization as traditional cgh. with the introduction of array cgh, the main limitation of conventional cgh, a low resolution, is overcome. in array cgh, the metaphase chromosomes are replaced by cloned dna fragments (+100–200 kb) of which the exact chromosomal location is known. this allows the detection of aberrations in more detail and, moreover, makes it possible to map the changes directly onto the genomic sequence. array cgh has proven to be a specific, sensitive, fast and highthroughput technique, with considerable advantages compared to other methods used for the analysis of dna copy number changes making it more amenable to diagnostic applications. using this method, copy number changes at a level of 5–10 kilobases of dna sequences can be detected. as of 2006, even high-resolution cgh (hr-cgh) arrays are accurate to detect structural variations (sv) at resolution of 200 bp. this method allows one to identify new recurrent chromosome changes such as microdeletions and duplications in human conditions such as cancer and birth defects due to chromosome aberrations. figure 2. array-cgh protocol methodology array cgh is based on the same principle as conventional cgh. in both techniques, dna from a reference (or control) sample and dna from a test (or patient) sample are differentially labelled with two different fluorophores and used as probes that are cohybridized competitively onto nucleic acid targets. in conventional cgh, the target is a reference metaphase spread. in array cgh, these targets can be genomic fragments cloned in a variety of vectors (such as bacs or plasmids), cdnas, or oligonucleotides. figure 2. is a schematic overview of the array cgh technique. dna from the sample to be tested is labeled with a red fluorophore (cyanine 5) and a reference dna sample is labeled with green fluorophore (cyanine 3). equal quantities of the two dna samples are mixed and cohybridized to a dna microarray of several thousand evenly spaced cloned dna fragments or oligonucleotides, which have been spotted in triplicate on the array. after hybridization, digital imaging systems are used to capture and quantify the relative fluorescence intensities of each of the hybridized fluorophores. the resulting ratio of the fluorescence intensities is proportional to the ratio of the copy numbers of dna sequences in the test and reference genomes. if the intensities of the flurochromes are equal on one probe, this region of the patient's genome is interpreted as having equal quantity of dna in the test and reference samples; if there is an altered cy3:cy5 ratio this indicates a loss or a gain of the patient dna at that specific genomic region. technological approaches to array cgh acgh profile of the imr32 neuroblastoma cell line array cgh has been implemented using a wide variety of techniques. therefore, some of the advantages and limitations of array cgh are dependent on the technique chosen. the initial approaches used arrays produced from large insert genomic dna clones, such as bacs. the use of bacs provides sufficient intense signals to detect single-copy changes and to locate aberration boundaries accurately. however, initial dna yields of isolated bac clones are low and dna amplification techniques are necessary. these techniques include ligation-mediated polymerase chain reaction (pcr), degenerate primer pcr using one or several sets of primers, and rolling circle amplification. arrays can also be constructed using cdna. 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. this results in the inability to detect single copy changes on a genome wide scale. the latest approach is spotting the arrays with short oligonucleotides. the amount of oligos is almost infinite, and the processing is rapid, cost-effective, and easy. although oligonucleotides do not have the sensitivity to detect single copy changes, averaging of ratios from oligos that map next to each other on the chromosome can compensate for the reduced sensitivity. it is also possible to use arrays which have overlapping probes so that specific breakpoints may be uncovered. design approaches there are two approaches to the design of microarrays for cgh applications: whole genome and targeted. whole genome arrays are designed to cover the entire human genome. they often include clones that provide an extensive coverage across the genome; and arrays that have contiguous coverage, within the limits of the genome. whole-genome arrays have been constructed mostly for research applications and have proven their outstanding worth in gene discovery. they are also very valuable in screening the genome for dna gains and losses at an unprecedented resolution. targeted arrays are designed for a specific region(s) of the genome for the purpose of evaluating that targeted segment. it may be designed to study a specific chromosome or chromosomal segment or to identify and evaluate specific dna dosage abnormalities in individuals with suspected microdeletion syndromes or subtelomeric rearrangements. the crucial goal of a targeted microarray in medical practice is to provide clinically useful results for diagnosis, genetic counseling, prognosis, and clinical management of unbalanced cytogenetic abnormalities. applications conventional 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. this approach can also be used to study chromosomal aberrations in fetal and neonatal genomes. furthermore, conventional cgh can be used in detecting chromosomal abnormalities and have been shown to be efficient in diagnosing complex abnormalities associated with human genetic disorders. in cancer research cgh data from several studies of the same tumor type show consistent patterns of non-random genetic aberrations. some of these changes appear to be common to various kinds of malignant tumors, while others are more tumor specific. 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. 3). other alterations, such as 12p and xp gains in testicular cancer, 13q gain 9q loss in bladder cancer, 14q loss in renal cancer and xp loss in ovarian cancer are more specific, and might reflect the unique selection forces operating during cancer development in different organs. array cgh is also frequently used in research and diagnostics of b cell malignancies, such as chronic lymphocytic leukemia. chromosomal aberrations cri du chat (cdc) is a syndrome caused by a partial deletion of the short arm of chromosome 5. several studies have shown that conventional cgh is suitable to detect the deletion, as well as more complex chromosomal alterations. for example, levy et al. (2002) reported an infant with a cat-like cry, the hallmark of cdc, but having an indistinct karyotype. cgh analysis revealed a loss of chromosomal material from 5p15.3 confirming the diagnosis clinically. 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. array cgh array cgh applications are mainly directed at detecting genomic abnormalities in cancer. however, array cgh is also suitable for the analysis of dna copy number aberrations that cause human genetic disorders. that is, array cgh is employed to uncover deletions, amplifications, breakpoints and ploidy abnormalities. earlier diagnosis is of benefit to the patient as they may undergo appropriate treatments and counseling to improve their prognosis. genomic abnormalities in cancer genetic alterations and rearrangements occur frequently in cancer and contribute to its pathogenesis. detecting these aberrations by array cgh provides information on the locations of important cancer genes and can have clinical use in diagnosis, cancer classification and prognostification. however, not all of the losses of genetic material are pathogenetic, since some dna material is physiologically lost during the rearrangement of immunoglobulin subgenes. 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. array cgh may also be applied not only to the discovery of chromosomal abnormalities in cancer, but also to the monitoring of the progression of tumors. differentiation between metastatic and mild lesions is also possible using fish once the abnormalities have been identified by array cgh. submicroscopic aberrations prader–willi syndrome (pws) is a paternal structural abnormality involving 15q11-13, while a maternal aberration in the same region causes angelman syndrome (as). in both syndromes, the majority of cases (75%) are the result of a 3–5 mb deletion of the pws/as critical region. these small aberrations cannot be detected using cytogenetics or conventional cgh, but can be readily detected using array cgh. 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. in all three cases, the abnormalities, ranging from 1.5 to 2.9mb, were readily identified. thus, array cgh was demonstrated to be a specific and sensitive approach in detecting submicroscopic aberrations. when using overlapping microarrays, it is also possible to uncover breakpoints involved in chromosomal aberrations. prenatal genetic diagnosis 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. it has the potential to detect cnvs and aneuploidy in eggs, sperm or embryos which may contribute to failure of the embryo to successfully implant, miscarriage or conditions such as down syndrome (trisomy 21). this makes array cgh a promising tool to reduce the incidence of life altering conditions and improve success rates of ivf attempts. the technique involves whole genome amplification from a single cell which is then used in the array cgh method. it may also be used in couples carrying chromosomal translocations such as balanced reciprocal translocations or robertsonian translocations, which have the potential to cause chromosomal imbalances in their offspring. limitations of cgh and array cgh a main disadvantage of conventional cgh is its inability to detect structural chromosomal aberrations without copy number changes, such as mosaicism, balanced chromosomal translocations, and inversions. cgh can also only detect gains and losses relative to the ploidy level. in addition, chromosomal regions with short repetitive dna sequences are highly variable between individuals and can interfere with cgh analysis. therefore, repetitive dna regions like centromeres and telomeres need to be blocked with unlabeled repetitive dna (e.g. cot1 dna) and/or can be omitted from screening. furthermore, the resolution of conventional cgh is a major practical problem that limits its clinical applications. 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. because of the limited resolution of metaphase chromosomes, aberrations smaller than 5–10 mb cannot be detected using conventional cgh. for the detection of such abnormalities, a high-resolution technique is required. array cgh overcomes many of these limitations. array cgh is characterized by a high resolution, its major advantage with respect to conventional cgh. 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. however, as in conventional cgh, the main disadvantage of array cgh is its inability to detect aberrations that do not result in copy number changes and is limited in its ability to detect mosaicism. the level of mosaicism that can be detected is dependent on the sensitivity and spatial resolution of the clones. at present, rearrangements present in approximately 50% of the cells is the detection limit. for the detection of such abnormalities, other techniques, such as sky (spectral karyotyping) or fish have to still be used.