Imagine trying to build a puzzle looking through a straw: You may see one piece but have no idea what others could possibly interact with that part of the puzzle. Due to the investment of time required to identify the presence of a given gene, genetic research occurred in this fashion, until recently... The development of microarray technology and its recent culmination in the gene chip allows researchers to monitor the presence of thousands of genes in a fraction of the time previously required. This same technology may soon be able to assay every gene present in a human’s genome in a matter of hours.
A microarray is designed to detect a very large set of specific genetic indicators in an organism. The arrays use messenger RNA (mRNA) as an indicator of a gene’s presence. In the process of turning the information encoded on a strand of DNA into a protein, mRNA is used to carry the DNA encoded blueprints for the protein from the nucleus to the cell's protein-constructing translational machinery. Therefore, every gene currently being expressed as a protein will have a piece of mRNA corresponding to it. Unfortunately, mRNA is very fragile and is only present in relatively small amounts inside of the cell. To avoid this problem, a reverse transcriptase is used to amplify and stabilize the signal. Reverse transcriptase takes mRNA and copies it many times over in the form of DNA, which is more stable. Retroviruses, like HIV, use reverse transcriptase to convert their RNA into DNA where it can then be inserted into and replicated along with the host’s chromosomes. DNA produced by the copying of mRNA using reverse transcriptase is called complementary DNA (cDNA). Microarrays detect the presence of the cDNA produced from the mRNA of a specific gene.
How Gene Chips Work
An array is specifically tailored to the needs of an investigation. A researcher can select hundreds of cDNAs from a massive library of the molecules. If scientists were to research the genetic basis for eye color in fruit flies, they would probably design an array containing the cDNA molecules corresponding to every gene known or suspected of relating to any variation of fruit fly eyes. Once the list of cDNA "genes" has been completed, the microarray can be built by permanently affixing each selected sample of cDNA into a specific grid of a polymer support. To screen an organism with the array, its mRNA is also converted into cDNA, which is then fluorescently labeled. The labeled cDNA is then washed over the array in a liquid medium. If a labeled cDNA is complementary to a cDNA on the array, it will attach to the fixed strand. The array can then be washed of any unattached molecules. Whatever grids fluoresce indicate that their corresponding genes are currently being expressed in the organism. The arrays produce a baffling amount of information for which there is no current means of finding all of the significant relationships.
Just as researchers scratched their heads at the massive amounts of information microarrays produced, Affymetrix unveiled the gene chip. The gene chip utilizes robotic technology for the fixing of microscopic cDNA samples onto the chip. Thousands of different samples may be fixed onto a single 2 cm2 chip. After the chip is prepared, Affymetrix exposes two separate samples of cDNA to the chip. The samples release different fluorescent colors red or green when excited with a laser. Wherever both samples sit on the same chip spot a yellow dot is produced. The application of two samples allows the immediate contrast between two different organisms. A fruit fly whose eyes are purple with yellow poky-dots can be sampled alongside a wild-type brown-eyed fruit fly. Any grid appearing as a green or red dot indicates a gene present in one fly that is not in the other and helps to quickly narrow down the genetic combination responsible for purple eyes with yellow poky-dots. The amount of each sample, which correlates to the amount of expression of a given gene, also produces colors of different intensities.
In 1997 researchers were able to find the exact combination of genes in a yeast cell in 51 hours, a procedure that once took years, with the use of a single gene chip. They simply ordered a chip with every gene known to exist in the yeast, collected their sample, and waited to see where the lights lit up. Correlative studies between organisms with differing traits have been made infinitely more accessible. For example, yeast used to make beer can only grow in up to 7% alcohol, while yeast used to make wine can tolerate up to 14% alcohol. Researchers could use the gene chip to make comparisons between the mRNA levels of the two species. Such comparisons could tell them which combinations of genes determine alcohol tolerance, and if desired, allow them to genetically alter the strains of yeast used for brewing beer to make beer with a much higher amount of alcohol.
Caveats
There are still several problems with the technology behind gene chips. One such problem is that they measure mRNA levels. This information is an indication of gene expression, and not necessarily an indicator of protein levels. Proteins are the key component for most investigations in that they are the end products of gene expression. There are many cellular controls other than gene expression to regulate protein levels or their effects, including increased degradation of specific mRNA (making the mRNA unavailable for translation), protein degradation, and modulation of enzyme activity. Furthermore, if the genes are not currently being expressed or being transcribed with mRNA during the period of analysis, then they will not be detectable with gene chip technology. Another problem with the technology is called transcription bias. Reverse transcriptase does not transcribe all cDNA molecules at the same rate. This difference would make the different intensities of fluorescence irrelevant in terms of comparing expression rates between two different gene. The most significant "problem" with this technology is a great one to have. It works so well there is currently too much information being generated. The solution is the development of strategies and software capable of dealing with sorting and compiling the data.
The improvements in chip design and scanning methods lead developers to believe up to four million genes can be represented on one 1 cm2 chip. The human genome contains about 100,000 genes. It may soon be possible to reasonably design studies comparing all of the genes expressed in breast cancer patients to those of healthy patients. The contrast would indicate exactly what genetic combinations are producing the cancer. Inhibitors of these gene proteins could be quickly immobilized to stop the disease in its tracks. The same process could quickly lead to gene therapy and cures to any genetically based disorder. The chip may even help to cure a bacterial disease. A gene chip could analyze a single swab of bacteria from a sore throat and instantly determine the medicines to which the bacteria have resistance. In the future, medicines could be administered according to how well patients having similar genes responded to the treatment. Lyonnaise des Eaux in Paris is even intending to install the chips in their water systems to tell instantly exactly what biological contaminants are in the water supply at any given time.
The introduction of this new biotechnology may have an unparalleled impact on the future of scientific research and the world around us. In a very brief period of time genetic research has gone from tunnel vision to panoramic views of an entire organism’s genome. Now we have to decide what to do, or not do, with it.
Gene Chips Web Site Links
Leming Shi - Great links and infromation on Gene Chips
