DNA microarray

From Academic Kids

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DNA_microarray.jpg
A DNA microarray, the different colours indicate relative expression of different genes.

A DNA microarray is a collection of microscopic DNA spots attached to a solid surface, such as glass, plastic or silicon chip forming an array. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously. The affixed DNA segments are known as probes, thousands of which can be used in a single DNA microarray. Microarray technology evolved from Southern Blotting, where fragmented DNA is attached to a substrate and then probed with a known gene or fragment. Measuring gene expression using microarrays is relevant to many areas of biology and medicine, such as studying treatments, disease and developmental stages.

Contents

Overview

Although the name GeneChip is a trademark of Affymetrix, this term is often used to refer to any microarray, not just those sold by Affymetrix. The term DNA chip, or simply chip, is also common. Developers of the technology use array to refer to microarrays in general, and reserve chip for reference to Affymetrix or CombiMatrix products. Affymetrix arrays use short oligonucleotide probes of 25 or fewer bases, and are often referred to as "high-density oligonucleotide microarrays. Other varieties of microarrays use as probes PCR products, genomic DNA, Bacterial artificial chromosomes, plasmids, or longer (35 to 70 base) oligonucleotides.

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 [1] (http://genomebiology.com/2004/5/8/R58), or electrochemistry on microelectrode arrays. The use of microarrays for expression profiling was first published in 1995 (Science) and the first complete eukaryotic genome (Saccharomyces cerevisiae) on a microarray was published in 1997 (Science).

The most common use of microarrays is to quantify mRNAs transcribed from different genes and which encode different proteins. RNA is extracted from many cells, ideally from a single cell type, then converted to cDNA or cRNA. The copies may be amplified by rtPCR. Fluorescent tags are enzymatically incorporated into the newly synthesized cDNA/cRNA or can be chemically attached to the new strands of DNA or RNA. A cDNA or cRNA molecule that contains a sequence complementary to one of the single-stranded probe sequences on the array will hybridize, via base pairing (more at DNA), to the spot at which the complementary probes are affixed. The spot will then fluoresce (or glow) when examined using a microarray scanner.

Increased or decreased fluorescence intensity indicates that cells in the sample have recently transcribed, or ceased transcription, of a gene that contains the probed sequence ("recently," because cells tend to degrade RNAs soon after transcription). The intensity of the fluorescence is roughly proportional to the number of copies of a particular mRNA that were present and thus roughly indicates the activity or expression level of that gene. Arrays can paint a picture or "profile" of which genes in the genome are active in a particular cell type and under a particular condition.

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Applications

Because many proteins have unknown functions, and because many genes are active all the time in all kinds of cells, researchers usually use microarrays to make comparisons between similar cell types. For example, an RNA sample from brain tumor cells, might be compared to a sample from healthy neurons or glia. Probes that bind RNA in the tumor sample but not in the healthy one may indicate genes that are uniquely associated with the disease. Typically in such a test, the two samples' cDNAs are tagged with two distinct colors, enabling comparison on a single chip. Researchers hope to find molecules that can be targeted for treatment with drugs among the various proteins encoded by disease-associated genes.

Although the chips detect RNAs that may or may not be translated into active proteins, scientists refer to these kinds of analysis as "expression analysis" or expression profiling. Since there are hundreds or thousands of distinct probes on an array, each microarray experiment can accomplish the equivalent of thousands of genetic tests in parallel. Arrays have therefore dramatically accelerated many types of investigations.

Microarrays are also being 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, chips 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 are also being evaluated by comparative genomic hybridization (CGH) arrays.

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 cancer.

Microarrays and bioinformatics

The lack of standardization in arrays presents an interoperability problem in bioinformatics, which hinders the exchange of array data. Many researchers use Affymetrix technology because it is popular and standardized, which can simplify the comparison of results from different laboratories. At the same time, various grass-roots open-source projects are attempting to facilitate the exchange and analysis of data produced with non-proprietary chips. The "Minimum Information About a Microarray Experiment" (MIAME) standard for describing a microarray experiment is being adopted by many journals as a requirement for the submission of papers incorporating microarray results.

External links

he:מערכי DNA ja:DNAマイクロアレイ pl:Mikromacierz vi:DNA microarray zh:生物晶片

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