|March 26, 2016|
A DNA microarray is a multiplex technology used in molecular biology. It consists of an arrayed series of thousands of microscopic spots of DNA oligonucleotides, called features, each containing picomole s (10???12 moles) of a specific DNA sequence, known as probe s (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.
Since an array can contain tens of thousands of probes, a microarray experiment can accomplish many genetic tests in parallel. Therefore arrays have dramatically accelerated many types of investigation.
In standard microarrays, the probes are attached via surface engineering to a solid surface by a covalent bond to a chemical matrix (via epoxy-silane, amino -silane, lysine, polyacrylamide or others). The solid surface can be glass or a silicon chip, in which case they are colloquially known as an Affy chip when an Affymetrix chip is used. Other microarray platforms, such as Illumina , use microscopic beads, instead of the large solid support. DNA arrays are different from other types of microarray only in that they either measure DNA or use DNA as part of its detection system.
DNA microarrays can be used to measure changes in expression levels, to detect single nucleotide polymorphisms (SNPs), or to genotype or resequence mutant genomes ( see uses and types section ). Microarrays also differ in fabrication, workings, accuracy, efficiency, and cost ( see fabrication section ). Additional factors for microarray experiments are the experimental design and the methods of analyzing the data ( see Bioinformatics section ).
Microarray technology evolved from Southern blotting, where fragmented DNA is attached to a substrate and then probed with a known gene or fragment. Nucleic Acids Res. 1992 Apr 11;20(7):1679-84.
Oligonucleotide hybridizations on glass supports: a novel linker for oligonucleotide synthesis and hybridization properties of oligonucleotides synthesised in situ.
Maskos U, Southern EM. 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 (Augenlicht and Kobrin, Cancer Research, 42, 1088-1093, 1982). 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 (Augenlicht et al, Cancer Research, 47, 6017-6021, 1987) and then to comparison of colonic tissues at different genetic risk (Augenlicht et al, Proceedings National Academy of Sciences, USA, 88, 3286-3289, 1991). 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.
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 hybridized. So fluorescently labelled 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 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.
An alternative to microarrays is serial analysis of gene expression, where the transcriptome is sequenced allowing an absolute measurement.
Many types of array exist and the broadest distinction is whether they are spatially arranged on 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.
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.
Spotted vs. in situ synthesised arrays
Microarrays can be fabricated using 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 oligonucleotide s, cDNA or small fragments of PCR products that correspond to mRNAs. The probes are synthesized prior to deposition on 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 is used by research scientists around the world to produce "in-house" printed microarrays from 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 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 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, 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 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 (Agilent and Affymetrix) on a silica substrate where light and light-sensitive masking agents are used to "build" a sequence one nucleotide at a time across the entire array.
Two-channel vs. one-channel detection
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.
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 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 , 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 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 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 so long as batch effects have been accounted for. A drawback to the one-color system is that, when compared to the two-color system, twice as many microarrays are needed to compare samples within an experiment.
The advent of inexpensive microarray experiments created several specific bioinformatics challenges:
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.
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 technical replicates may be two independent RNA extractions or 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.
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:
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:
Microarray data may require further processing aimed at reducing 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 replicate s; 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.
Relation between probe and gene
The relation between a probe and the mRNA that it is expected to detect is problematic. On the one hand, 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. On the other hand, 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 was found to be more useful when compared to other similar datasets. The sheer volume (in bytes), specialized formats (such as MIAME), and curation efforts associated with the datasets require specialized databases to store the data.
GNU Free Documentation License. It uses material from the Wikipedia article "DNA microarrays".
All informatin on the site is © www.diseases-diagnosis.com 2002-2011. Last revised: January 2, 2011|
Are you interested in our site or/and want to use our information? please read how to contact us and our copyrights.
To let us provide you with high quality information, you can help us by making a more or less donation: