Editing Comparative genomic hybridization (section)

==Array comparative genomic hybridization==
{{anchor|Array-CGH}}
Array comparative genomic hybridization (also microarray-based comparative genomic hybridization, matrix CGH, array CGH, aCGH) is a molecular [[cytogenetic]] technique for the detection of chromosomal [[Copy-number variation|copy number changes]] on a genome wide and high-resolution scale.<ref>{{cite journal | vauthors = Pinkel D, Albertson DG | year = 2005 | title = Array comparative genomic hybridization and its applications in cancer | journal = Nat Genet | volume = 37 | issue = 6s| pages = 11–17 | doi=10.1038/ng1569| pmid = 15920524 | doi-access = free }}</ref> Array CGH compares the patient's genome against a reference genome and identifies differences between the two genomes, and hence locates regions of [[genetic imbalance|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 [[molecular cloning|cloned]] DNA fragments (+100–200 kb) of which the exact chromosomal location is known. This allows the detection of [[chromosome abnormality|aberrations]] in more detail and, moreover, makes it possible to map the changes directly onto the genomic sequence.<ref name="Oostlander,Meijer,Ylstra">Oostlander AE, Meijer GA, Ylstra B (2004) Microarray-based comparative genomic hybridization and its applications in human genetics. Clin Genet 66:488–495.</ref>

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. Using this method, [[copy number]] changes at a level of 5–10 [[kilobase]]s of DNA sequences can be detected.<ref>{{cite journal|vauthors=Ren H, Francis W, Boys A, Chueh AC, Wong N, La P, Wong LH, Ryan J, Slater HR, Choo KH |title=BAC-based PCR fragment microarray: high-resolution detection of chromosomal deletion and duplication breakpoints |journal=Human Mutation |pmid=15832308 |doi=10.1002/humu.20164 |volume=25 |issue=5 |date=May 2005 |pages=476–82|s2cid=28030180 |doi-access=free }}</ref> {{As of|2006}}, even [[high-resolution CGH]] ([[HR-CGH]]) arrays are accurate to detect [[structural variation]]s (SV) at resolution of 200 bp.<ref>{{cite journal |vauthors=Urban AE, Korbel JO, Selzer R, Richmond T, Hacker A, Popescu GV, Cubells JF, Green R, Emanuel BS, Gerstein MB, Weissman SM, Snyder M |title=High-resolution mapping of DNA copy alterations in human chromosome 22 using high-density tiling oligonucleotide arrays |journal=Proc Natl Acad Sci U S A |date=21 March 2006 |volume=103 |issue=12 |pages=4534–4539 |pmid=16537408 |pmc=1450206 |doi=10.1073/pnas.0511340103|bibcode=2006PNAS..103.4534U |doi-access=free }}</ref> This method allows one to identify new recurrent chromosome changes such as [[microdeletion]]s and duplications in human conditions such as [[cancer]] and [[birth defect]]s due to chromosome aberrations.

[[File:Array-CGH protocol.svg|right|thumb|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 [[hybridization probe|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 [[Bacterial artificial chromosome|BACs]] or [[plasmids]]), [[Complementary DNA|cDNAs]], or [[oligonucleotides]].<ref name="Bejjani,Shaffer">Bejjani BA, Shaffer LG (2006) Applications of array-based comparative genomic hybridization to clinical diagnostics. J Mol Diagn 8:528–533.</ref>

Figure 2.<ref name="Oostlander,Meijer,Ylstra" /> 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.<ref name="Bejjani,Shaffer" /> 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.<ref>Shinawi M, Cheung SW (2008) The array CGH and its clinical applications. Drug Discovery Today 13:760–769.</ref>

===Technological approaches to array CGH===

[[File:ACGH profile of the IMR32 neuroblastoma cell line.svg|right|thumb|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 [[bacterial artificial chromosome|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 [[Covalent bond|ligation]]-mediated polymerase chain reaction (PCR), degenerate primer PCR using one or several sets of primers, and [[rolling circle amplification]].<ref>{{cite journal | vauthors = Fiegler H, Carr P, Douglas EJ, Burford DC, Hunt S, Scott CE, Smith J, Vetrie D, Gorman P, Tomlinson IP, Carter NP | year = 2003 | title = DNA microarrays for comparative genomic hybridization based on DOP-PCR amplification of BAC and PAC clones | journal = Genes Chromosomes Cancer | volume = 36 | issue = 4| pages = 361–374 | doi=10.1002/gcc.10155| pmid = 12619160 | s2cid = 6929961 }}</ref> 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.<ref name="Oostlander,Meijer,Ylstra" /> This results in the inability to detect single copy changes on a genome wide scale.<ref>{{cite journal | vauthors = Pollack JR, Perou CM, Alizadeh AA, Eisen MB, Pergamenschikov A, Williams CF, Jeffrey SS, Botstein D, Brown PO | year = 1999 | title = Genome-wide analysis of DNA copy number changes using cDNA microarrays | doi = 10.1038/12640 | pmid = 10471496 | journal = Nat Genet | volume = 23 | issue = 1| pages = 41–46 | s2cid = 997032 }}</ref> 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.<ref>{{cite journal | vauthors = Carvalho B, Ouwerkerk E, Meijer GA, Ylstra B | year = 2004 | title = High resolution microarray comparative genomic hybridization analysis using spotted oligonucleotides | journal = J Clin Pathol | volume = 57 | issue = 6| pages = 644–646 | doi=10.1136/jcp.2003.013029| pmid = 15166273 | pmc = 1770328 }}</ref> 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.<ref name="Bejjani,Shaffer" />

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.<ref name="Bejjani,Shaffer" />