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DNA microarray
A DNA microarray (also commonly known as gene chip, DNA chip, or biochip) is a collection of microscopic DNA spots attached to 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 (1012 moles) of a specific DNA sequence, known as probes (or reporters). These can be a short section of a gene or other DNA element that are used to hybridize a cDNA or cRNA sample (called target) under high-stringency conditions. Probe-target hybridization 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.
History
Microarray technology evolved from Southern blotting, where fragmented DNA is attached to a substrate and then probed with a known gene or fragment.[1] The first reported use of this approach was the analysis of 378 arrayed lysed bacterial colonies each harboring a different sequence which were assayed in multiple replicas for expression of the genes in multiple normal and tumor tissue.[2] This was expanded to analysis of more than 4000 human sequences with computer driven scanning and image processing for quantitative analysis of the sequences in human colonic tumors and normal tissue [3] and then to comparison of colonic tissues at different genetic risk.[4] The use of a collection of distinct DNAs in arrays for expression profiling was also described in 1987, and the arrayed DNAs were used to identify genes whose expression is modulated by interferon.[5] These early gene arrays were made by spotting cDNAs onto filter paper with a pin-spotting device. The use of miniaturized microarrays for gene expression profiling was first reported in 1995,[6] and a complete eukaryotic genome (Saccharomyces cerevisiae) on a microarray was published in 1997.[7]
DNA microarray
Principle
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 a nucleotide sequence means tighter non-covalent bonding between the two strands. After washing off of non-specific bonding sequences, only strongly paired strands will remain Hybridization of the target to the probe. hybridized. So fluorescently labeled target sequences that bind to a probe sequence generate a signal that depends on the strength of the hybridization determined by the number of paired bases, the hybridization conditions (such as temperature), and washing after hybridization. Total strength of the signal, from a spot (feature), depends upon the amount of target sample binding to the probes present on that spot. Microarrays use relative quantization 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.
Two Affymetrix chips. A match is shown at bottom left for size comparison.
DNA microarray 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:
Application or technology Gene expression profiling Synopsis
In an mRNA or gene expression profiling experiment the expression levels of thousands of genes are simultaneously monitored to study the effects of certain treatments, diseases, and developmental stages on gene expression. For example, microarray-based gene expression profiling can be used to identify genes whose expression is changed in response to [8] pathogens or other organisms by comparing gene expression in infected to that in uninfected cells or tissues. Assessing genome content in different cells or closely related organisms. [9][10]
Small microarrays to check IDs of organisms in food and feed (like GMO [11]), mycoplasms in cell culture, or pathogens for disease detection, mostly combining PCR and microarray technology.
Chromatin DNA sequences bound to a particular protein can be isolated by immunoprecipitating that protein (ChIP), these fragments immunoprecipitation on can be then hybridized to a microarray (such as a tiling array) allowing the determination of protein binding site Chip occupancy throughout the genome. Example protein to immunoprecipitate are histone modifications (H3K27me3, H3K4me2, H3K9me3, etc.), Polycomb-group protein (PRC2:Suz12, PRC1:YY1) and trithorax-group protein (Ash1) to study the epigenetic landscape or RNA Polymerase II to study the transcription landscape. DamID Analogously to ChIP, genomic regions bound by a protein of interest can be isolated and used to probe a microarray to determine binding site occupancy. Unlike ChIP, DamID does not require antibodies but makes use of adenine methylation near the protein's binding sites to selectively amplify those regions, introduced by expressing minute amounts of protein of interest fused to bacterial DNA adenine methyltransferase. [12] Identifying single nucleotide polymorphism among alleles within or between populations. Several applications of microarrays make use of SNP detection, including Genotyping, forensic analysis, measuring predisposition to disease, identifying drug-candidates, evaluating germline mutations in individuals or somatic mutations in cancers, assessing loss of heterozygosity, or genetic linkage analysis. An 'exon junction array design uses probes specific to the expected or potential splice sites of predicted exons for a gene. It is of intermediate density, or coverage, to a typical gene expression array (with 1-3 probes per gene) and a genomic tiling array (with hundreds or thousands of probes per gene). It is used to assay the expression of alternative splice forms of a gene. Exon arrays have a different design, employing probes designed to detect each individual exon for known or predicted genes, and can be used for detecting different splicing isoforms. A Fusion gene microarray can detect fusion transcripts, e.g. from cancer specimens. The principle behind this is building on the alternative splicing microarrays. The oligo design strategy enables combined measurements of chimeric transcript junctions with exon-wise measurements of individual fusion partners. Genome tiling arrays consist of overlapping probes designed to densely represent a genomic region of interest, sometimes as large as an entire human chromosome. The purpose is to empirically detect expression of transcripts or alternatively splice forms which may not have been previously known or predicted.
SNP detection
Tiling array
DNA microarray
Fabrication
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 may have as few as 10 probes or up to 2.1 million micrometre-scale probes from commercial vendors.
DNA microarray
DNA microarray
Experimental design
Due to the biological complexity of gene expression, 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.
Gene expression values from microarray experiments can be represented as heat maps to visualize the result of data analysis.
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 (two RNA samples obtained from each experimental unit) help to ensure precision and allow for testing differences within treatment groups. The biological replicates include independent RNA extractions and technical replicates may be two aliquots of 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.[20]
Standardization
Microarray data 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 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" 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.[21] The MGED Society has developed standards for the representation of gene expression experiment results and relevant annotations.
DNA microarray
Statistical analysis
Microarray data sets are commonly very large, and analytical precision 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: Image analysis: gridding, spot recognition of the scanned image (segmentation algorithm), removal or marking of poor-quality and low-intensity features (called flagging). Data processing: background subtraction (based on global or local background), determination of spot intensities and intensity ratios, visualisation of data (e.g. see MA plot), and log-transformation of ratios, global or local normalization of intensity ratios, and segmentation into different copy number regions using step detection algorithms.[22] Identification of statistically significant changes: t-test, ANOVA, Bayesian method[23] MannWhitney test methods tailored to microarray data sets, which take into account multiple comparisons[24] or cluster analysis.[25] These methods assess statistical power based on the variation present in the data and the number of experimental replicates, and can help minimize Type I and type II errors in the analyses.[26] Network-based methods: Statistical methods that take the underlying structure of gene networks into account, representing either associative or causative interactions or dependencies among gene products.[27] Microarray data may require further processing aimed at reducing the dimensionality of the data to aid comprehension and more focused analysis.[28] 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.[29]
Data warehousing
Microarray data 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.
References
[1] Maskos, U; Southern, EM (11 Apr 1992). "Oligonucleotide hybridizations on glass supports: a novel linker for oligonucleotide synthesis and hybridization properties of oligonucleotides synthesised in situ". Nucleic Acids Res. (Maskos U, Southern EM.) 20 (7): 167984. doi:10.1093/nar/20.7.1679. PMC312256. PMID1579459. [2] Augenlicht LH, Kobrin D (1982). "Cloning and screening of sequences expressed in a mouse colon tumor" (http:/ / cancerres. aacrjournals. org/ content/ 42/ 3/ 1088. long). Cancer Research 42 (3): 10881093. PMID7059971. . [3] Augenlicht et al.; Wahrman, MZ; Halsey, H; Anderson, L; Taylor, J; Lipkin, M (1987). "Expression of cloned sequences in biopsies of human colonic tissue and in colonic carcinoma cells induced to differentiate in vitro". Cancer Research 47 (22): 60176021. PMID3664505. [4] Augenlicht et al. (1991). "Patterns of Gene Expression that Characterize the Colonic Mucosa in Patients at Genetic Risk for Colonic Cancer". Proceedings National Academy of Sciences 88 (8): 32863289. doi:10.1073/pnas.88.8.3286. [5] Kulesh DA, Clive DR, Zarlenga DS, Greene JJ (1987). "Identification of interferon-modulated proliferation-related cDNA sequences". Proc Natl Acad Sci USA 84 (23): 84538457. doi:10.1073/pnas.84.23.8453. PMC299562. PMID2446323. [6] Schena M, Shalon D, Davis RW, Brown PO (1995). "Quantitative monitoring of gene expression patterns with a complementary DNA microarray". Science 270 (5235): 467470. doi:10.1126/science.270.5235.467. PMID7569999.
DNA microarray
[7] Lashkari DA, DeRisi JL, McCusker JH, Namath AF, Gentile C, Hwang SY, Brown PO, Davis RW (1997). "Yeast microarrays for genome wide parallel genetic and gene expression analysis". Proc Natl Acad Sci USA 94 (24): 1305713062. doi:10.1073/pnas.94.24.13057. PMC24262. PMID9371799. [8] Adomas A, Heller G, Olson A, Osborne J, Karlsson M, Nahalkova J, Van Zyl L, Sederoff R, Stenlid J, Finlay R, Asiegbu FO (2008). "Comparative analysis of transcript abundance in Pinus sylvestris after challenge with a saprotrophic, pathogenic or mutualistic fungus". Tree Physiol. 28 (6): 885897. PMID18381269. [9] Pollack JR, Perou CM, Alizadeh AA, Eisen MB, Pergamenschikov A, Williams CF, Jeffrey SS, Botstein D, Brown PO (1999). "Genome-wide analysis of DNA copy-number changes using cDNA microarrays". Nat Genet 23 (1): 4146. doi:10.1038/14385. PMID10471496. [10] Moran G, Stokes C, Thewes S, Hube B, Coleman DC, Sullivan D (2004). "Comparative genomics using Candida albicans DNA microarrays reveals absence and divergence of virulence-associated genes in Candida dubliniensis". Microbiology 150 (Pt 10): 33633382. doi:10.1099/mic.0.27221-0. PMID15470115. [11] http:/ / bgmo. jrc. ec. europa. eu/ home/ docs. htm [12] Hacia JG, Fan JB, Ryder O, Jin L, Edgemon K, Ghandour G, Mayer RA, Sun B, Hsie L, Robbins CM, Brody LC, Wang D, Lander ES, Lipshutz R, Fodor SP, Collins FS (1999). "Determination of ancestral alleles for human single-nucleotide polymorphisms using high-density oligonucleotide arrays". Nat Genet 22 (2): 164167. doi:10.1038/9674. PMID10369258. [13] J Biochem Biophys Methods. 2000 Mar 16;42(3):105-10. DNA-printing: utilization of a standard inkjet printer for the transfer of nucleic acids to solid supports. Goldmann T, Gonzalez JS. [14] Lausted C et al. (2004). "POSaM: a fast, flexible, open-source, inkjet oligonucleotide synthesizer and microarrayer" (http:/ / genomebiology. com/ 2004/ 5/ 8/ R58). Genome Biology 5 (8): R58. doi:10.1186/gb-2004-5-8-r58. PMC507883. PMID15287980. . [15] Bammler T, Beyer RP; Consortium, Members of the Toxicogenomics Research; Kerr, X; Jing, LX; Lapidus, S; Lasarev, DA; Paules, RS; Li, JL et al (2005). "Standardizing global gene expression analysis between laboratories and across platforms". Nat Methods 2 (5): 351356. doi:10.1038/nmeth0605-477a. PMID15846362. [16] Pease AC, Solas D, Sullivan EJ, Cronin MT, Holmes CP, Fodor SP. (1994). "Light-generated oligonucleotide arrays for rapid DNA sequence analysis". PNAS 91 (11): 50225026. doi:10.1073/pnas.91.11.5022. PMC43922. PMID8197176. [17] Nuwaysir EF, Huang W, Albert TJ, Singh J, Nuwaysir K, Pitas A, Richmond T, Gorski T, Berg JP, Ballin J, McCormick M, Norton J, Pollock T, Sumwalt T, Butcher L, Porter D, Molla M, Hall C, Blattner F, Sussman MR, Wallace RL, Cerrina F, Green RD. (2002). "Gene Expression Analysis Using Oligonucleotide Arrays Produced by Maskless Photolithography". Genome Res 12 (11): 17491755. doi:10.1101/gr.362402. PMC187555. PMID12421762. [18] Shalon D, Smith SJ, Brown PO (1996). "A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization". Genome Res 6 (7): 639645. doi:10.1101/gr.6.7.639. PMID8796352. [19] Tang T, Franois N, Glatigny A, Agier N, Mucchielli MH, Aggerbeck L, Delacroix H (2007). "Expression ratio evaluation in two-colour microarray experiments is significantly improved by correcting image misalignment". Bioinformatics 23 (20): 26862691. doi:10.1093/bioinformatics/btm399. PMID17698492. [20] Churchill, GA (2002). "Fundamentals of experimental design for cDNA microarrays" (http:/ / www. vmrf. org/ research-websites/ gcf/ Forms/ Churchill. pdf) ( Scholar search (http:/ / scholar. google. co. uk/ scholar?hl=en& lr=& q=intitle:Fundamentals+ of+ experimental+ design+ for+ cDNA+ microarrays& as_publication=Nature+ genetics+ suppliment& as_ylo=2002& as_yhi=2002& btnG=Search)). 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[Available at http:/ / www. biomedcentral. com/ 1471-2105/ 8/ 111 "Evaluation of gene-expression clustering via mutual information distance measure"]. BMC Bioinformatics 8 (1): 111. doi:10.1186/1471-2105-8-111. PMC1858704. PMID17397530. Available at . [26] Wei C, Li J, Bumgarner RE. (2004). "Sample size for detecting differentially expressed genes in microarray experiments". BMC Genomics 5: 87. doi:10.1186/1471-2164-5-87. PMC533874. PMID15533245. [27] Emmert-Streib, F. and Dehmer, M. (2008). Analysis of Microarray Data A Network-Based Approach. Wiley-VCH. ISBN3-527-31822-4. [28] Wouters L, Ghlmann HW, Bijnens L, Kass SU, Molenberghs G, Lewi PJ (2003). "Graphical exploration of gene expression data: a comparative study of three multivariate methods". Biometrics 59 (4): 11311139. doi:10.1111/j.0006-341X.2003.00130.x. PMID14969494. [29] Jain N, Thatte J, Braciale T, Ley K, O'Connell M, Lee JK (2003). "Local-pooled-error test for identifying differentially expressed genes with a small number of replicated microarrays". Bioinformatics 19 (15): 19451951. doi:10.1093/bioinformatics/btg264. PMID14555628.
DNA microarray
Glossary
An Array or slide is a collection of features spatially arranged in a two dimensional grid, arranged in columns and rows. Block or subarray: a group of spots, typically made in one print round; several subarrays/blocks form an array. Case/control: an experimental design paradigm especially suited to the two-colour array system, in which a condition chosen as control (such as healthy tissue or state) is compared to an altered condition (such as a diseased tissue or state). Channel: the fluorescence output recorded in the scanner for an individual fluorophore and can even be ultraviolet. Dye flip or Dye swap or Fluor reversal: reciprocal labelling of DNA targets with the two dyes to account for dye bias in experiments. Scanner: an instrument used to detect and quantify the intensity of fluorescence of spots on a microarray slide, by selectively exciting fluorophores with a laser and measuring the fluorescence with a filter (optics) photomultiplier system. Spot or feature: a small area on an array slide that contains picomoles of specific DNA samples. For other relevant terms see: Glossary of gene expression terms Protocol (natural sciences)
External links
Many important links can be found at the Open Directory Project Gene Expression (http://www.dmoz.org/Science/Biology/Biochemistry_and_Molecular_Biology/ Gene_Expression/) at the Open Directory Project Micro Scale Products and Services for Biochemistry and Molecular Biology (http://www.dmoz.org/ Science/Biology/Biochemistry_and_Molecular_Biology/Products_and_Services/Micro_Scale/) at the Open Directory Project Products and Services for Gene Expression (http://www.dmoz.org/Science/Biology/ Biochemistry_and_Molecular_Biology/Gene_Expression/Products_and_Services/) at the Open Directory Project Online Services for Gene Expression Analysis (http://www.dmoz.org/Science/Biology/Bioinformatics/ Online_Services/Gene_Expression_and_Regulation/) at the Open Directory Project PLoS Biology Primer: Microarray Analysis (http://biology.plosjournals.org/perlserv/?request=get-document& doi=10.1371/journal.pbio.0000015) Rundown of microarray technology (http://www.genome.gov/page.cfm?pageID=10000533) ArrayMining.net (http://www.arraymining.net) - a free web-server for online microarray analysis CLASSIFI (http://pathcuric1.swmed.edu/pathdb/classifi.html) - Gene Ontology-based gene cluster classification resource Microarray - How does it work? (http://www.unsolvedmysteries.oregonstate.edu/microarray_07) What Are DNA Microarrays (http://www.bioinformaticstutorials.com/?p=8) - A Non-Biologists Introduction to Microarrays Microarray data processing using Self-Organizing Maps tutorial: Part 1 (http://blog.peltarion.com/2007/04/ 10/the-self-organized-gene-part-1) Part 2 (http://blog.peltarion.com/2007/06/13/ the-self-organized-gene-part-2)
PNAS Commentary: Discovery of Principles of Nature from Mathematical Modeling of DNA Microarray Data (http://www.pnas.org/content/103/44/16063.extract)
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