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| molecular biology | |
| gene expression | |
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A DNA microarray (also commonly known as '' Gene or Genome chip'', ''DNA chip'', or ''gene array'') is a collection of microscopic DNA spots, commonly representing single Gene s, arrayed on a solid surface by Covalent attachment to chemically suitable matrices. 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. Qualitative or quantitative measurements with DNA microarrays utilize the selective nature of DNA-DNA or DNA-RNA Hybridization under high-stringency conditions and Fluorophore -based detection. DNA arrays are commonly used for Expression Profiling , i.e., monitoring Expression levels of thousands of genes simultaneously, or for comparative genomic hybridization. INTRODUCTION 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. Applications of these arrays include:
FABRICATION Microarrays can be fabricated using a variety of technologies, including printing with fine-pointed pins onto glass slides, on microelectrode arrays. DNA microarrays can be used to detect RNAs that may or may not be translated into active proteins. Scientists refer to this kind of analysis as "expression Analysis" or Expression Profiling . Since there can be tens of thousands of distinct probes on an array, each microarray experiment can accomplish the equivalent number of genetic tests in parallel. Arrays have therefore dramatically accelerated many types of investigations. The use of microarrays for gene expression profiling was first published in 1995Schena M, Shalon D, Davis RW, Brown PO. (1995). Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science. Oct 20; 270 (5235): 467-70. ('' Science '') and the first complete Eukaryotic genome ('' Saccharomyces Cerevisiae '') on a microarray was published in 1997 (''Science''). Spotted microarrays In spotted microarrays (or '''two-channel or two-colour microarrays'''), the probes are Oligonucleotide s, CDNA or small fragments of PCR products that correspond to MRNA s and are spotted onto the microarray surface. This type of array is typically Hybridized with cDNA from two samples to be compared (e.g. diseased tissue versus healthy tissue) that are labeled with two different Fluorophores (e.g. Rhodamine (Cyanine 5, green) and Fluorescein (Cyanine 3, red)). The two samples are mixed and hybridized to a single microarray that is then scanned in a microarray scanner to visualize Fluorescence of the two fluorophores. Relative intensities of each fluorophore are then used to identify up-regulated and down-regulated genes in ratio-based analysis. Absolute levels of gene expression cannot be determined in the two-colour array, but relative differences in expression among different spots (=genes) can be estimated with some oligonucleotide arrays. Examples of providers for such microarrays includes Agilent with their Dual-Mode platform, Eppendorf with their DualChip platform, and TeleChem International with ArrayIt . Oligonucleotide microarrays In oligonucleotide microarrays (or '''single-channel microarrays'''), the probes are designed to match parts of the sequence of known or predicted mRNAs. There are commercially available designs that cover complete genomes from companies such as . These microarrays give estimations of the absolute value of gene expression and therefore the comparison of two conditions requires the use of two separate microarrays. Oligonucleotide Arrays can be either produced by piezoelectric deposition with full length oligonucleotides or in-situ synthesis. Long Oligonucleotide Arrays are composed of 60-mers, or 50-mers and are produced by ink-jet printing on a silica substrate. Short Oligonucleotide Arrays are composed of 25-mer or 30-mer and are produced by photolithographic synthesis (Affymetrix) on a silica substrate or piezoelectric deposition (GE Healthcare) on an acrylamide matrix. More recently, Maskless Array Synthesis from NimbleGen Systems has combined flexibility with large numbers of probes. Arrays can contain up to 390,000 spots, from a custom array design. New array formats are being developed to study specific pathways or disease states for a systems biology approach. Oligonucleotide microarrays often contain control probes designed to hybridize with RNA Spike-in s. The degree of hybridization between the spike-ins and the control probes is used to normalize the hybridization measurements for the target probes. GENOTYPING MICROARRAYS See Also: SNP array DNA microarrays can also be used to ''read'' the sequence of a genome in particular positions. SNP microarrays are a particular type of DNA microarrays that are used to identify genetic variation in individuals and across populations. Short oligonucleotide arrays can be used to identify the Single Nucleotide Polymorphisms (SNPs) that are thought to be responsible for genetic variation and the source of susceptibility to genetically caused diseases. Generally termed Genotyping applications, DNA microarrays may be used in this fashion for forensic applications, rapidly discovering or measuring genetic predisposition to disease, or identifying DNA-based drug candidates. These SNP microarrays are also being used to profile Somatic Mutations in Cancer , specifically Loss Of Heterozygosity events and amplifications and deletions of regions of DNA. Amplifications and deletions can also be detected using Comparative Genomic Hybridization , or aCGH, in conjunction with microarrays, but may be limited in detecting novel Copy Number Polymorphisms , or CNPs, by probe coverage. Resequencing arrays have also been developed to sequence portions of the Genome in individuals. These arrays may be used to evaluate Germline mutations in individuals, or somatic mutations in cancers. Genome tiling arrays include overlapping oligonucleotides designed to blanket an entire genomic region of interest. Many companies have successfully designed tiling arrays that cover whole human chromosomes. MICROARRAYS AND BIOINFORMATICS s to visualize the result of data analysis.]] 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.
Standardization The lack of standardization in arrays presents an Interoperability problem in Bioinformatics , which hinders the exchange of array data. Various grass-roots Open-source projects are attempting to facilitate the exchange and analysis of data produced with non-proprietary chips.
Statistical analysis The analysis of DNA microarrays poses a large number of Statistical problems, including the Normalization of the data. There are dozens of proposed normalization methods in the published literature; as in many other cases where authorities disagree, a sound conservative approach is to try a number of popular normalization methods and compare the conclusions reached: how sensitive are the main conclusions to the method chosen? |
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