A DNA microarray is an arrayed series of microscopic spots of DNA oligonucleotides, called features, where each feature contains picomoles of a specific unique sequence, such as a stretch of a gene sequence, which is used to measure the relative abundance of a sequence between samples, either differentially labelled or on different microarrays.
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These molecules are the used as probes to which only the correct target sequence will hybridise under high-stringency conditions; where the target is the labelled mix of either RNA or DNA to be analysed, in the majority of cases with a fluorophore-based detection system.
In "traditional" microarrays the probes are bound to a solid surface by covalent attachment to a chemical matrix. The solid surface can either be glass or a silicon chip, in which case they are commonly known as gene,genome chip or colloquially Affy Chip when an Affymetrix chip is used. Some recent platforms, such as Illumina, use microscopic beads, instead of the large solid support (glass or treated silicon) present in traditional microarrays. 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 arrays are commonly used for expression profiling, i.e., monitoring expression levels of thousands of genes simultaneously, or for comparative genomic hybridization.Types of Array
Arrays of DNA can either be spatially arranged, as in the commonly known gene or genome chip, DNA chip, or gene array, or can be specific DNA sequences tagged or labelled such that they can be independently identified in solution. The traditional solid-phase array is a collection of microscopic DNA spots attached to a solid surface, such as glass, plastic or silicon chip. The affixed DNA segments are known as probes (although some sources will use different nomenclature such as reporters), thousands of which can be placed in known locations on a single DNA microarray. Microarray technology evolved from Southern blotting, whereby fragmented DNA is attached to a substrate and then probed with a known gene or fragment. DNA microarrays can be used to detect DNA (e.g., in comparative genomic hybridization); it also permits detection of RNA (most commonly as cDNA after reverse transcription) that may or may not be translated into proteins, which is referred to as "expression analysis" or expression profiling.
Since there can be tens of thousands of distinct probes on an array, each microarray experiment can potentially accomplish the equivalent number of genetic tests in parallel. Arrays have therefore dramatically accelerated many types of investigations. The use of a collection of distinct DNAs in arrays for expression profiling was first described in 1987, and the arrayed DNAs were used to identify genes whose expression is modulated by interferon. 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, and a complete eukaryotic genome (Saccharomyces cerevisiae) on a microarray was published in 1997. Fabrication Spotted vs. oligonucleotide 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 oligonucleotides, 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 is 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. Applied Microarrays offers a commercial array platform called the "CodeLink" system where 30-mer oligonucleotide probes (sequences of 30 nucleotides in length) are piezoelectrically deposited on an acrylamide matrix without any contact being made between the depositing equipment and the array surface itself. These arrays are comparable in quality to most manufactured arrays and generally superior to in-house printed 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. 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 vs. one-color 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. Fluorescent dyes commonly used for cDNA labelling include Cy3, which has a fluorescence emission wavelength of 570 nm (corresponding to the green part of the light spectrum), and Cy5 with a fluorescence emission wavelength of 670 nm (corresponding to the red part of the light spectrum). The two Cy-labelled 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 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 contain 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, 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 fluorescence labeling, and TeleChem International with Arrayit.
In single-channel microarrays or one-color microarrays, the arrays are designed to give estimations of the absolute levels of gene expression. Therefore the comparison of two conditions requires two separate single-dye hybridizations. As only a single dye is used, the data collected represent absolute values of gene expression. These may be compared to other genes within a sample or to reference "normalizing" probes used to calibrate data across the entire array and across multiple arrays. Three popular single-channel systems are the Affymetrix "Gene Chip", 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; the absolute values of gene expression may be compared between studies conducted months or years apart. 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.