microarrays.html

DNA Microarrays

This page was created as an assignment for an undergraduate class at Davidson College

DNA microarrays are a new molecular tool with the potential to revolutionize diagnostics, gene identification, and the study of gene expression patterns. The basis of microarrays is a chip, similar to a computer microchip, only it is labeled with oligonucleotides that are capable of binding to complementary strands of DNA. In the past genes could only be examined one at a time. DNA chips, however, allow researchers to analyze huge numbers of genes all at once. The ability to examine patterns of gene expression allows much greater insight into the function of gene pathways instead of just the function of single genes.

DNA chips were first introduced in 1991 by Affymetrix, a drug-discovery company. They synthesize their chips by binding nucleotides to a silicon surface coated with linker molecules. They selectively bind nucleotides to certain areas of the chip at a time by using a "blocking" compound that caps the linkers until a light is shone through a mask "uncapping" the desired areas. By using different masks, they can create an array of oligonucleotides in a specific pattern on each chip. In just 32 cycles of this masking/binding process, a chip can be created that contains over 65,000 oligonucleotides that are all eight base pairs long. A chip can then be used as a probe for thousands of different sequences all at once. Fluroescently labeled cDNA can be floated over the chips, allowed to bind, then examined with a laser to see where the DNA has bound. Each oligo sequence on the chip is known and, therefore, tells the sequence of the complementary DNA that has bound to each location on the chip.

Figure 1. DNA chips are synthesized using silicon squares labeled with oligonucleotides. Oligonucleotides are added to the chips using linker molecules which are capped with a "blocking" compound. A light is shone through a mask onto certain areas of the chip, uncapping the areas so that oligonucleotides can be added. The exposed areas are then recapped and the process is repeated using a mask that allows a different pattern of light to shine onto the chip, exposing a different area. Adapted from <http://www.affymetrix.com/technology/synthesis.html>

Figure 2. A. GeneChip® expression arrays simultaneously and quantitatively interrogate thousands of mRNA transcripts (genes or ESTs), simplifying large genomic studies. B. Each transcript is represented on a probe array by multiple probe pairs so that you can differentiate among closely related members of gene families. C. Each probe cell contains millions of copies of a specific oligonucleotide probe, permitting the accurate and sensitive detection of low-intensity mRNA hybridization patterns. Image and figure legend adapted from <http://www.affymetrix.com/products/expression.shtml>

Other companies have developed variations on the Affymetrix chips. Hyseq, Inc. developed chips tagged with fluorescently labeled known oligos. When these are mixed with an unknown DNA sample, a fluorescent spot appears everywhere that the DNA binds, and the sequence of that DNA can be detected based on the known oligo sequence to which it bound. A computer is used to piece together the partial DNA sequences after the binding process is repeated several times.

Incyte Pharmaceuticals has created a glass chip that has an array of gene fragments that are much longer (500 to 5000 base pairs). Messenger RNA can be isolated from two tissue samples, for example, one normal sample and one diseased sample or one sample treated with a drug. Each RNA sample is labeled with a different color fluorescent tag and then mixed with the same chip. When the chip is then scanned for the two different colors, the gene expression patterns of each sample can be compared.

Figure 3. Glass DNA chips can be used to examine differential gene expression between to tissue samples simultaneously using a different colored fluorescent tag for each sample. Image adapted from <http://gem.incyte.com/gem/technology/scan.shtml>

Nanogen researchers have discovered a way to speed up the time required to allow the oligos to bind to their complementary DNA. The known oligos are maneuvered on the chip using an electric field. This allows the oligos to "go out and get" their complementary DNA rather than "wait" passively for it to float by and bind.

Figure 4. An electric field is applied to these chips allowing DNA fragments to base pair to complementary oligos in minutes. Image adapted from <http://www.nanogen.com/tech.htm>

Another electronic method is being used by Clinical Microsensors, who have abandoned the use of fluorescent tags for detecting the location of DNA fragments on the chips. They use a separate probe that carries iron and will attach to DNA that has bound to its complementary oligo on the chip. The oligos are attached on a grid of electrodes that can detect the presence of the iron and indicates the position of the bound DNA.

Figure 5. Microarrays are built on a grid of electrodes. A separate probe containing iron (which can be detected by the electrodes) assists in identifying DNA that has bound to its matching oligo. Image adapted from <http://www.microsensor.com/chemical surface.htm>

DNA microarrays have countless applications in the world of genomics, diagnostics, and pharmaceuticals. These chips have been used for diverse purposes in recent research. DNA microarrays were used to compare different strains of bacteria that cause tuberculosis in order to better understand the evolution of these bacterial strains, an understanding that will lead to better preventative and diagnostic care for tuberculosis (Behr et al., 1999). Iyer et al. (1999) used DNA microarrays to give a temporal account of human fibroblast activity at the level of gene expression in response to fetal bovine serum (FBS), a provider of growth factors, and suggest that fibroblasts play an even larger role in the physiology of wound repair than was previously thought. A study of the genome of Saccharomyces cerevisiae using DNA microarrays allowed researchers to scan, map, and analyze allelic variation between two strains of yeast without performing time consuming tasks such as PCR or creating new DNA constructs (Winzeler et al., 1998).

In the future, DNA chips could be used to make commonplace diagnostic techniques such as diagnosing strep throat even cheaper, faster, and more accurate. In the meantime, these nine square centimeter chips have already lead to the discover of PLA2, a protein produced by prostate cancer cells (and not healthy prostate cells), a protein called melastatin, which is produced by melanoma cancer cells, and mutations in BRCA1, the familiar breast cancer gene.