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87-597: CGH may refer to: Comparative genomic hybridization Computer-generated holography the IATA airport code of Congonhas-São Paulo Airport Changi General Hospital , a hospital in Simei, Singapore Colorado General Hospital, former name of University of Colorado Hospital cGh physics , a characterization of unified physical theories encompassing relativity, gravitation and quantum mechanics Coventry Godiva Harriers Chief of

174-405: A laser beam of a defined wavelength. Relative intensities of each fluorophore may then be used in ratio-based analysis to identify up-regulated and down-regulated genes. Oligonucleotide microarrays often carry control probes designed to hybridize with RNA spike-ins . The degree of hybridization between the spike-ins and the control probes is used to normalize the hybridization measurements for

261-476: 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 applications are mainly directed at detecting genomic abnormalities in cancer. However, array CGH

348-471: A coverslip. A fluorescence microscope with the appropriate filters for the DAPI stain as well as the two fluorophores utilised is required for visualisation, and these filters should also minimise the crosstalk between the fluorophores, such as narrow band pass filters. The microscope must provide uniform illumination without chromatic variation, be appropriately aligned and have a "plan" type of objective which

435-431: A diagram identifying chromosomes based on banding patterns. Interpretation of the ratio profiles is conducted either using fixed or statistical thresholds ( confidence intervals ). When using confidence intervals, gains or losses are identified when 95% of the fluorescence ratio does not contain 1.0. Extreme care must be taken to avoid contamination of any step involving DNA, especially with the test DNA as contamination of

522-425: A fluorescence emission wavelength of 570 nm (corresponding to the green part of the light spectrum), and Cy 5 with a fluorescence emission wavelength of 670 nm (corresponding to the red part of the light spectrum). The two Cy-labeled cDNA samples are mixed and hybridized to a single microarray that is then scanned in a microarray scanner to visualize fluorescence of the two fluorophores after excitation with

609-405: 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,

696-460: 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

783-400: 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% CO 2 . Colchicine

870-472: 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 high-throughput technique, with considerable advantages compared to other methods used for

957-409: A nucleotide sequence means tighter non-covalent bonding between the two strands. After washing off non-specific bonding sequences, only strongly paired strands will remain hybridized. Fluorescently labeled target sequences that bind to a probe sequence generate a signal that depends on the hybridization conditions (such as temperature), and washing after hybridization. Total strength of the signal, from

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1044-440: 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

1131-592: 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. Standard phenol extraction

1218-548: 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. 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

1305-525: A solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome. Each DNA spot contains picomoles (10 moles ) of a specific DNA sequence, known as probes (or reporters or oligos ). These can be a short section of a gene or other DNA element that are used to hybridize a cDNA or cRNA (also called anti-sense RNA) sample (called target ) under high-stringency conditions. Probe-target hybridization

1392-566: 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. Conventional CGH has been used mainly for

1479-413: A spot (feature), depends upon the amount of target sample binding to the probes present on that spot. Microarrays use relative quantitation in which the intensity of a feature is compared to the intensity of the same feature under a different condition, and the identity of the feature is known by its position. Many types of arrays exist and the broadest distinction is whether they are spatially arranged on

1566-496: A surface or on coded beads: DNA microarrays can be used to detect DNA (as in comparative genomic hybridization ), or detect RNA (most commonly as cDNA after reverse transcription ) that may or may not be translated into proteins. The process of measuring gene expression via cDNA is called expression analysis or expression profiling . Applications include: Specialised arrays tailored to particular crops are becoming increasingly popular in molecular breeding applications. In

1653-782: A time across the entire array. Each applicable probe is selectively "unmasked" prior to bathing the array in a solution of a single nucleotide, then a masking reaction takes place and the next set of probes are unmasked in preparation for a different nucleotide exposure. After many repetitions, the sequences of every probe become fully constructed. More recently, Maskless Array Synthesis from NimbleGen Systems has combined flexibility with large numbers of probes. Two-color microarrays or two-channel microarrays are typically hybridized with cDNA prepared from two samples to be compared (e.g. diseased tissue versus healthy tissue) and that are labeled with two different fluorophores . Fluorescent dyes commonly used for cDNA labeling include Cy 3, which has

1740-434: 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

1827-435: A variety of technologies, including printing with fine-pointed pins onto glass slides, photolithography using pre-made masks, photolithography using dynamic micromirror devices, ink-jet printing, or electrochemistry on microelectrode arrays. In spotted microarrays , the probes are oligonucleotides , cDNA or small fragments of PCR products that correspond to mRNAs . The probes are synthesized prior to deposition on

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1914-509: 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

2001-404: Is apochromatic and give a magnification of x63 or x100. The image should be recorded using a camera with spatial resolution at least 0.1 μm at the specimen level and give an image of at least 600x600 pixels. The camera must also be able to integrate the image for at least 5 to 10 seconds, with a minimum photometric resolution of 8 bit. Dedicated CGH software is commercially available for

2088-422: 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

2175-511: 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. Genetic alterations and rearrangements occur frequently in cancer and contribute to its pathogenesis. Detecting these aberrations by array CGH provides information on

2262-400: 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. The DNA on the slide is a reference sample, and is thus obtained from

2349-427: 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

2436-446: 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

2523-492: Is being conducted by the US Food and Drug Administration (FDA) to develop standards and quality control metrics which will eventually allow the use of MicroArray data in drug discovery, clinical practice and regulatory decision-making. The MGED Society has developed standards for the representation of gene expression experiment results and relevant annotations. Microarray data sets are commonly very large, and analytical precision

2610-481: 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

2697-439: 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. DNA microarrays A DNA microarray (also commonly known as DNA chip or biochip ) is a collection of microscopic DNA spots attached to

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2784-504: Is different from Wikidata All article disambiguation pages All disambiguation pages Comparative genomic hybridization 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 (fluorescent molecules) of different colours (usually red and green), denaturation of

2871-427: Is difficult to exchange due to the lack of standardization in platform fabrication, assay protocols, and analysis methods. This presents an interoperability problem in bioinformatics . Various grass-roots open-source projects are trying to ease the exchange and analysis of data produced with non-proprietary chips: For example, the "Minimum Information About a Microarray Experiment" ( MIAME ) checklist helps define

2958-406: Is expected to detect is not trivial. Some mRNAs may cross-hybridize probes in the array that are supposed to detect another mRNA. In addition, mRNAs may experience amplification bias that is sequence or molecule-specific. Thirdly, probes that are designed to detect the mRNA of a particular gene may be relying on genomic EST information that is incorrectly associated with that gene. Microarray data

3045-545: Is in SNPs arrays for polymorphisms in cardiovascular diseases, cancer, pathogens and GWAS analysis. It is also used for the identification of structural variations and the measurement of gene expression. The core principle behind microarrays is hybridization between two DNA strands, the property of complementary nucleic acid sequences to specifically pair with each other by forming hydrogen bonds between complementary nucleotide base pairs . A high number of complementary base pairs in

3132-419: Is influenced by a number of variables. Statistical challenges include taking into account effects of background noise and appropriate normalization of the data. Normalization methods may be suited to specific platforms and, in the case of commercial platforms, the analysis may be proprietary. Algorithms that affect statistical analysis include: Microarray data may require further processing aimed at reducing

3219-956: 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. 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

3306-447: 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. Unlabelled Life Technologies Corporation's Cot-1 DNA (placental DNA enriched with repetitive sequences of length 50bp-100bp)is added to block normal repetitive DNA sequences, particularly at centromeres and telomeres , as these sequences, if detected, may reduce

3393-496: 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 (0.35 μg/ml), for chromosome identification, and sealing with

3480-414: Is used by research scientists around the world to produce "in-house" printed microarrays in their own labs. These arrays may be easily customized for each experiment, because researchers can choose the probes and printing locations on the arrays, synthesize the probes in their own lab (or collaborating facility), and spot the arrays. They can then generate their own labeled samples for hybridization, hybridize

3567-459: Is used to obtain DNA from test or reference (karyotypically normal individual) 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

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3654-416: Is usually detected and quantified by detection of fluorophore -, silver-, or chemiluminescence -labeled targets to determine relative abundance of nucleic acid sequences in the target. The original nucleic acid arrays were macro arrays approximately 9 cm × 12 cm and the first computerized image based analysis was published in 1981. It was invented by Patrick O. Brown . An example of its application

3741-620: 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

3828-525: 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. Nick translation is used to label the DNA and involves cutting DNA and substituting nucleotides labelled with fluorophores (direct labelling) or biotin or oxigenin to have fluophore conjugated antibodies added later (indirect labelling). It

3915-437: 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

4002-416: 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

4089-748: The Order of the Golden Heart of Kenya Camiguin General Hospital , a hospital in Mambajao, Philippines Topics referred to by the same term [REDACTED] This disambiguation page lists articles associated with the title CGH . If an internal link led you here, you may wish to change the link to point directly to the intended article. Retrieved from " https://en.wikipedia.org/w/index.php?title=CGH&oldid=1157398188 " Category : Disambiguation pages Hidden categories: Short description

4176-418: 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. Though not yet a widely employed technique, the use of array CGH as a tool for preimplantation genetic screening

4263-527: The aetiology of known and unknown conditions to be discovered. 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

4350-544: 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. Array CGH

4437-479: The array surface and are then "spotted" onto glass. A common approach utilizes an array of fine pins or needles controlled by a robotic arm that is dipped into wells containing DNA probes and then depositing each probe at designated locations on the array surface. The resulting "grid" of probes represents the nucleic acid profiles of the prepared probes and is ready to receive complementary cDNA or cRNA "targets" derived from experimental or clinical samples. This technique

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4524-419: 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

4611-430: The arrays provide intensity data for each probe or probe set indicating a relative level of hybridization with the labeled target. However, they do not truly indicate abundance levels of a gene but rather relative abundance when compared to other samples or conditions when processed in the same experiment. Each RNA molecule encounters protocol and batch-specific bias during amplification, labeling, and hybridization phases of

4698-627: The considerations of experimental design that are discussed in the expression profiling article are of critical importance if statistically and biologically valid conclusions are to be drawn from the data. There are three main elements to consider when designing a microarray experiment. First, replication of the biological samples is essential for drawing conclusions from the experiment. Second, technical replicates (e.g. two RNA samples obtained from each experimental unit) may help to quantitate precision. The biological replicates include independent RNA extractions. Technical replicates may be two aliquots of

4785-399: The desired purpose; longer probes are more specific to individual target genes, shorter probes may be spotted in higher density across the array and are cheaper to manufacture. One technique used to produce oligonucleotide arrays include photolithographic synthesis (Affymetrix) on a silica substrate where light and light-sensitive masking agents are used to "build" a sequence one nucleotide at

4872-473: The dimensionality of the data to aid comprehension and more focused analysis. Other methods permit analysis of data consisting of a low number of biological or technical replicates ; for example, the Local Pooled Error (LPE) test pools standard deviations of genes with similar expression levels in an effort to compensate for insufficient replication. The relation between a probe and the mRNA that it

4959-438: 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. 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,

5046-458: 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

5133-584: The experiment making comparisons between genes for the same microarray uninformative. The comparison of two conditions for the same gene requires two separate single-dye hybridizations. Several popular single-channel systems are the Affymetrix "Gene Chip", Illumina "Bead Chip", Agilent single-channel arrays, the Applied Microarrays "CodeLink" arrays, and the Eppendorf "DualChip & Silverquant". One strength of

5220-431: The fluorescence ratio and cause gains or losses to escape detection. 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 (SSC) solution at

5307-489: The future they could be used to screen seedlings at early stages to lower the number of unneeded seedlings tried out in breeding operations. Microarrays can be manufactured in different ways, depending on the number of probes under examination, costs, customization requirements, and the type of scientific question being asked. Arrays from commercial vendors may have as few as 10 probes or as many as 5 million or more micrometre-scale probes. Microarrays can be fabricated using

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5394-474: 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. CGH data from several studies of

5481-454: The image processing step, and is required to subtract background noise, remove and segment materials not of chromosomal origin, normalize the fluorescence ratio, carry out interactive karyotyping and chromosome scaling to standard length. A "relative copy number karyotype" which presents chromosomal areas of deletions or amplifications is generated by averaging the ratios of a number of high quality metaphases and plotting them along an ideogram,

5568-447: The level of detail that should exist and is being adopted by many journals as a requirement for the submission of papers incorporating microarray results. But MIAME does not describe the format for the information, so while many formats can support the MIAME requirements, as of 2007 no format permits verification of complete semantic compliance. The "MicroArray Quality Control (MAQC) Project"

5655-583: 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

5742-496: 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,

5829-409: 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 . This improved technique allows for

5916-430: The multiple levels of replication in experimental design ( Experimental design ); the number of platforms and independent groups and data format ( Standardization ); the statistical treatment of the data ( Data analysis ); mapping each probe to the mRNA transcript that it measures ( Annotation ); the sheer volume of data and the ability to share it ( Data warehousing ). Due to the biological complexity of gene expression,

6003-617: The only choice in some situations. Suppose i {\displaystyle i} samples need to be compared: then the number of experiments required using the two channel arrays quickly becomes unfeasible, unless a sample is used as a reference. two channel microarray (with reference) This is an example of a DNA microarray experiment which includes details for a particular case to better explain DNA microarray experiments, while listing modifications for RNA or other alternative experiments. The advent of inexpensive microarray experiments created several specific bioinformatics challenges:

6090-480: 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

6177-447: The same extraction. Third, spots of each cDNA clone or oligonucleotide are present as replicates (at least duplicates) on the microarray slide, to provide a measure of technical precision in each hybridization. It is critical that information about the sample preparation and handling is discussed, in order to help identify the independent units in the experiment and to avoid inflated estimates of statistical significance . Microarray data

6264-434: The same level of sensitivity compared to commercial oligonucleotide arrays, possibly owing to the small batch sizes and reduced printing efficiencies when compared to industrial manufactures of oligo arrays. In oligonucleotide microarrays , the probes are short sequences designed to match parts of the sequence of known or predicted open reading frames . Although oligonucleotide probes are often used in "spotted" microarrays,

6351-545: 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

6438-604: 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

6525-408: The sample with normal DNA will skew results closer to 1.0, thus abnormalities may go undetected. FISH, PCR and flow cytometry experiments may be employed to confirm results. 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 changes on

6612-405: The samples to the array, and finally scan the arrays with their own equipment. This provides a relatively low-cost microarray that may be customized for each study, and avoids the costs of purchasing often more expensive commercial arrays that may represent vast numbers of genes that are not of interest to the investigator. Publications exist which indicate in-house spotted microarrays may not provide

6699-429: 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. There are two approaches to the design of microarrays for CGH applications: whole genome and targeted. Whole genome arrays are designed to cover

6786-512: The single-dye system lies in the fact that an aberrant sample cannot affect the raw data derived from other samples, because each array chip is exposed to only one sample (as opposed to a two-color system in which a single low-quality sample may drastically impinge on overall data precision even if the other sample was of high quality). Another benefit is that data are more easily compared to arrays from different experiments as long as batch effects have been accounted for. One channel microarray may be

6873-568: The target probes. Although absolute levels of gene expression may be determined in the two-color array in rare instances, the relative differences in expression among different spots within a sample and between samples is the preferred method of data analysis for the two-color system. Examples of providers for such microarrays includes Agilent with their Dual-Mode platform, Eppendorf with their DualChip platform for colorimetric Silverquant labeling, and TeleChem International with Arrayit . In single-channel microarrays or one-color microarrays ,

6960-529: The term "oligonucleotide array" most often refers to a specific technique of manufacturing. Oligonucleotide arrays are produced by printing short oligonucleotide sequences designed to represent a single gene or family of gene splice-variants by synthesizing this sequence directly onto the array surface instead of depositing intact sequences. Sequences may be longer (60-mer probes such as the Agilent design) or shorter (25-mer probes produced by Affymetrix ) depending on

7047-447: 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

7134-415: 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

7221-499: 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. 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

7308-445: 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,

7395-449: 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

7482-488: Was found to be more useful when compared to other similar datasets. The sheer volume of data, specialized formats (such as MIAME ), and curation efforts associated with the datasets require specialized databases to store the data. A number of open-source data warehousing solutions, such as InterMine and BioMart , have been created for the specific purpose of integrating diverse biological datasets, and also support analysis. Advances in massively parallel sequencing has led to

7569-603: 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 (BACs) can be used on DNA microarrays which may contain up to 2 million probes. Array CGH

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