It is widely believed that thousands of genes and their products (i.e., RNA and proteins) in a given living organism function in a complicated and orchestrated way that creates the mystery of life. However, traditional methods in molecular biology generally work on a "one gene in one experiment" basis, which means that the throughput is very limited and the "whole picture" of gene function is hard to obtain.
In the past few years, as a result of the Human Genome Project, there has been an explosion in the amount of information available about the DNA sequence of the human genome. Consequently, researchers have identified a large number of novel genes within these previously unknown sequences. The challenge currently facing scientists is to find a way to organize and catalog this vast amount of information into a usable form. Only after the functions of the new genes are discovered will the full impact of the Human Genome Project be realized, so that researchers can have a better picture of the interactions among thousands of genes simultaneously.
The microarrays theory relies on the following theory - every cell of the body contains a full set of chromosomes and identical genes (with only few exceptional cells). Only a fraction of these genes are turned on, however, and it is the subset that is "expressed" that confers unique properties to each cell type.
"Gene expression" is the term used to describe the transcription of the information contained within the DNA, the repository of genetic information, into messenger RNA (mRNA) molecules that are then translated into the proteins that perform most of the critical functions of cells. Scientists study the kinds and amounts of mRNA produced by a cell to learn which genes are expressed, which in turn provides insights into how the cell responds to its changing needs. Gene expression is a highly complex and tightly regulated process that allows a cell to respond dynamically both to environmental stimuli and to its own changing needs. This mechanism acts as both an "on/off" switch to control which genes are expressed in a cell as well as a "volume control" that increases or decreases the level of expression of particular genes as necessary.
Initially developed to enhance genomic sequencing projects, especially the Human Genome Project, DNA chips are finding applications throughout the field of molecular biology. Gene scanning techniques that are based on oligonucleotide arrays called DNA chips, provide a rapid method to analyze thousands of genes simultaneously. DNA chips are thus potentially very powerful tools for gaining insight into the complexities of gene expression, detecting genetic variations, making new gene discoveries, fingerprinting and developing new diagnostic tools.
The production of DNA chips have evolved along two major pathways: one method uses nucleic acids that have been immobilized on the chip surface sequentially to form oligonucleotides and the other method involves complementary DNA from an individual with a known genetic mutation as a source of prefabricated oligonucleotides. In either case, the problem lies with how to attach the nucleic acids or cDNA to the chip.
Chips using nucleic acids are produced using photolithography. Photolithography, according to the Science article by Stephen Fodor, consists of the modification of synthetic linkers, containing photochemically removable protecting groups, attached to a glass substrate, usually a silicon-derivative glass chip. Light is directed at the photolithographic "mask" at specific areas of the chip in order to facilitate the removal of the photoactive groups, yielding 5( hydroxy groups. These modified groups are now capable of binding other nucleotides, generating a highly specific probe, which contains the sequence of a known disease causing genetic mutation.
The other method, described in the DNA Chips and Microassays website, uses purified single-stranded cDNA from an individual with a known genetic disease, requiring the use of touch or fine micropipetting, to spot the cDNA onto the surface of the chip. The cDNA immobilizes on the chip through covalent bonds, due to the positively charged surface, produced by amino silane or polylysine. For both types of chips, a potential DNA target sequence, from an asymptomatic individual, is fluorescently tagged and allowed to interact with the probes. Hybridization will occur at complementary sequences between the two samples resulting in a fluorescent image, which is then scanned by a laser beam and analyzed by a computer. The intensity of fluorescent light varies with the strength of the hybridization, thus providing a quantitative 'snapshot' of gene expression.
This approach, requiring only minute consumption of chemical reagents and minute preparations of biological samples, can scan more than 400,000 probes placed on a single chip measuring 1.28cm X 1.28cm in size. As of now, specific chips are available for as little as $100, but could cost over thousands of dollars, once custom-made chips are available. In the future, attempts to design chips using the computer, instead of doing it by hand, will greatly speed up the process allowing companies to make custom chips in one day, as opposed to months, which would lower the cost of production. Consequently, DNA chips could probably sell for about $50, providing access to scientists regardless of their funding situation.
About Author / Additional Info:
Trending Articles ( Receiving maximum views in the last few days )