When physiological changes occur, some genes tend to become more active while other genes become inactive. In most biological processes, more than one gene is involved. In order to understand the functioning of an organism, scientists have examined many of an organism's life process simultaneously to understand how they respond to various changes over time.
DNA microarrays, also called genome chips, biochips, DNA chips, or gene arrays, technology has incredibly improved the throughput in genetic and biological experimentation. The basic idea behind this technology is that genes and gene products (proteins, RNA) work together in a complex way to enable an organism function as a whole. DNA microarray technology has allowed scientists to adopt the approach called "whole picture" in biological experimentation. This means, the total sum of all interaction taking place across a full set of gene sequences are measured simultaneously and calculated instantly by an ordered array.
The DNA microarray technology provides a universal and powerful tool for researchers in exploring the gene in a comprehensive and systematic fashion to survey DNA and RNA variations. The basic principle underlying this technology is the affinity and specificity of complementary base pairs. DNA microarrays are used in the exploration and exploitation of gene properties in many ways. Gene attributes that are of immense interest include those that are cordial to microarray approach and its transcription, genotype, products subcellular location, mutant phenotypes, and translation.
The experimental methods involved in exploring the gene attributes vary in complexity. Studying differential expression at mRNA level is quite straightforward. While, measuring a DNA microarray's differential hybridization to fluorescently labeled cDNAs that are prepared from two mRNA samples are used in comparing the abundance of mRNA from each gene.
Technically, the first microarray to be developed was called the Southern blot, where nucleic acid molecules (labeled) were used in interrogating nucleic acid molecules that were immobilized on a solid support. Today, thanks to the advancement in biotechnology, researchers have developed variant forms of DNA microarrays. The two common and popular design formats are:
Developed by the Affymetrix, Inc. this format was called the DNA Chip Technology. An oligonucleotides array (10- to 80-mer oligos) and probes of peptide nucleic acid are synthesized either by conventional methods or in situ and deposited on the chip. To complementary sequences, the labeled unknown DNA sample is applied to the chip for hybridization. Then, the abundance and identity of the complementary sequences are determined.
Stanford University Format
Pioneered by researchers at the University of Stanford, this format is called the DNA Microarray Technology. Probes of DNA between the range of 500 and 5000 bp long are made immobile on a non-porous material (example, glass) via robotic spotting. An unknown single-stranded sequence or a mixture of many target sequences is poured over the plate. The plate is later rinsed and the spots where probe complements are found are then visualized.
Fabrication of Microarrays
DNA microarrays are nothing but ordered sets of known DNA sequences. Arrays are usually rectangular shaped. A DNA spot is usually less than 200 microns in diameter and is placed exactly at a desired location. Each DNA spot has a specific sequence. The test material has RNA amplified by PCR methodology. Microarrays consist of certain key components including:
• Media or Material
• A Spotter
• Labeling and Detection and
• Analytical Software
Majority of the microarrays come in standard microscope slide configuration (for example, CMT-GAPS amino-salanized slides or polylysine-coated glass). Usually, labeling and detection are done based on fluorescence systems. Each DNA probe is labeled with a colored fluor, which is visibly different, such that it is distinguished by reading devices that are fitted with optical filters. Readers, also called scanners, use either white light of high intensity or laser-induced fluorescence. The three common fabrication technologies include:
• Photolithographic Fabrication: This technology uses semi-conductor technology in chip fabrication
• Piezoelectric Fabrication: An adaption of the ink-jet printing technology
• Micro-spotting: This technology is cordial to oligos and biomolecules
DNA microarrays are expensive with the scanner and the scanner alone costs round $60,000. This price is expected to decrease steadily over the time as more researchers have begun using the technology and manufacturers of the equipments are forced to compete for customers. As an alternative, in the future, researchers may purchase pre-fabricated microarrays of genes of interest.
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