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Within a year we will know virtually the entire sequence of the human genome—the genetic instructions that specify the molecular components, the design, and the operating software for the human body. This knowledge will transform medicine, giving us the means to see and to understand human anatomy, specialization, physiology, and pathophysiology in molecular detail. The genomes of more than 30 frequently studied organisms, including many human pathogens, have already been fully sequenced, and almost half of the sequence of the human genome is currently available in fragmentary form in public databases (NCBI GeneMap at http://www.ncbi.nlm.nih.gov/genemap). Not only will this new knowledge open a molecular window on a largely unexplored world of human biology, but it will also provide a way to see and to understand the molecular scripts that guide normal physiology and development and their alterations in disease. Here we focus on the use of DNA microarrays, or DNA chips as "microscopes," to observe the physiology of the living genome.
The Basics of DNA Microarrays
DNA microarrays are microscopic, physically ordered arrays of thousands of DNAs of known sequences, attached to solid surfaces. All of the genes in a genome can be arrayed in an area no larger than a standard microscope slide. Today, the largest DNA microarrays contain elements representing almost 40,000 genes, roughly half of the predicted number of genes in the human genome. A few years from now, when we know the complete catalog of human genes, DNA microarrays will allow us to watch every gene in our genomes.
To survey the expression of genes, RNA transcripts are isolated from cells, labeled with a fluorescent dye, and hybridized to a DNA microarray (Figure 1). During this hybridization process, the DNA sequences of the immobilized elements "capture" their complementary cognates in the fluorescent probe mixture. The fluorescent signal at the "spot" in the array representing each individual gene provides a quantitative readout of the level of expression of that gene in the sample.1 This straightforward procedure provides a systematic, quantitative way to monitor expression of tens of thousands of genes simultaneously.
Use of DNA Microarray to Detect Differences in Gene Expression in Normal vs Malignant Breast Epithelial Cells.
Messenger RNA from normal breast tissue is labeled with green fluorescent dye and messenger RNA from the malignant breast tissue is labeled with red fluorescent dye using a reverse transcription reaction. The resulting fluorescent complementary DNAs from the 2 samples are then combined and hybridized to the DNA microarray. The relative abundance of different genes in the 2 samples is reflected by the color of the corresponding spots in the microarray and can be quantitated using a scanning laser microscope. In the example shown, the microarray spot denoting ErbB2 fluoresces red, indicating that the oncogene ErbB2 is expressed at abnormally high levels in the malignant breast tumor cells.
Each cell in the human body expresses a specific set of genes according to a precisely controlled program that gives each cell its distinctive design and functional capabilities. Cells further employ signal transduction systems to collect information about their condition, including the presence of infection, stress, drugs, injury, growth factors or hormones, and convert these inputs to changes in gene expression. The gene expression patterns thus reflect a cell's internal state and microenvironment, creating a molecular "picture" of the cell's state. Since DNA microarrays detect gene expression patterns, they can be used to capture these molecular pictures and thus to deduce the condition of cells.
Because the expression pattern of a gene is closely tied to its biological role, systematic microarray studies of global gene expression can provide remarkably detailed clues to the functions of specific genes. This is an important advance, since we currently know the functions of fewer than 5% of the genes in the human genome.
A broad range of clinical applications has been suggested for DNA microarrays, and many have already been demonstrated in recent studies. These include applications such as messenger RNA expression profiling for improved disease classification,2,3 genotyping of polymorphisms affecting disease susceptibility,4 identification of genetic lesions within malignancies,5,6 design and discovery of therapeutics, and sequencing of DNA.7
Gene Expression Profiling
Most disease processes are accompanied not only by characteristic macroscopic or histological changes but also by systematic changes in gene expression patterns. For some pathological processes, such as cancer, inappropriate gene expression is fundamental to pathogenesis. For others, the gene expression programs, both in cells directly affected by a disease and in healthy cells responding to the local and systemic effects of a disease, can give us a detailed molecular picture of the pathogenic process.
Subtle but critically important molecular differences that have heretofore gone unrecognized enable us to distinguish superficially similar disease processes that differ importantly in their natural histories and therapeutic responses. We expect that the detailed molecular pictures provided by genomic expression analysis will revolutionize molecular medicine just as high-resolution radiographic imaging methods have revolutionized diagnosis and treatment at the gross anatomic level.
Several studies have used gene expression signatures captured using DNA microarrays for the molecular classification of cancer. One study recently demonstrated the ability of these profiles to distinguish distinct pathological entities, such as acute myeloid leukemia and acute lymphoblastic leukemia, on the basis of their distinctive gene expression programs.3 More promisingly, DNA microarrays have revealed distinct new diseases. For example, a recent study showed that diffuse large B-cell lymphoma, the most common non-Hodgkin lymphoma, is actually comprised of at least 2 distinct diseases with distinct expression profiles and strikingly different clinical courses.2 Because discrete disease variants will often require different therapies, the ability to classify diseases on the basis of gene expression profiles will undoubtedly improve management of many disorders.
A common strategy in the development of new therapeutics is to screen candidate compounds for activity against disease-specific cellular targets. However, this approach has been limited by the scarcity of known molecular targets. Microarray-based gene expression analyses will facilitate the rapid identification of disease-specific genes and reveal the cellular pathways involved in pathophysiology. The discovery of disease-specific genes and pathways has immediate implications for drug development. In the simplest scenario, genes overexpressed in diseased cells (such as the Her-2/neu in breast cancers) could serve as potential drug targets. In addition, established drugs that act through unknown molecular mechanisms can be studied using DNA microarrays. The gene expression responses of cells exposed to these agents should help elucidate their mechanisms of action and facilitate the development of new drugs with similar specificities.8
Other applications of DNA microarrays include pharmacogenomic methods for improved drug development and measurements of DNA variation associated with pathogenesis or involved in disease predisposition. See the full-length Web version of this article online at http://www.msjama.org for an in-depth discussion of these and related microarray applications.
Beyond Nucleic Acids
The ability to use a DNA sequence directly as a reagent for detecting and assaying copies of that sequence in a biological sample has been exploited in the first wave of genomic assays and diagnostics. Genome sequences also provide a less immediate but equally valuable route to assays for the protein products of every gene. We are thus on the threshold of a formidable new challenge and opportunity: discovering the biological activities of proteins on a genomic scale. This rapidly expanding enterprise has been termed "proteomics." Its tools include diverse mass spectroscopic methods,9 antibody microarrays, which simultaneously assay the presence or absence of multiple disease-marker proteins within bodily fluids, and genetic and "chemical genetic" technologies.1011 Such tools, combined with the continued use of DNA microarrays, will have an immense impact on clinical diagnostics and therapeutics in coming years.
Diehn M, Alizadeh AA, Brown PO. Examining the Living Genome in Health and Disease With DNA Microarrays. JAMA. 2000;283(17):2298–2299. doi:10.1001/jama.283.17.2298-JMS0503-4-1
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