From The Microscope To Microarrays

XI ANNUAL CONFERENCE, IAMM AP CHAPTER : FEBRYUARY 2008, MEDICITI, HYDERABAD

FROM THE MICROSCOPE TO MICROARRAYS Dr D.S.Murty, Assistant Professor, Department of Microbiology, S V Medical College, Tirupati. Email : [email protected]

In the present era of genomics, there has been an explosion in the amount of information available about the DNA. With the completion of the draft sequence of the human genome, the attention has now been focused on the ways in which the emergent technologies will have direct applications to patient care. DNA Microarrays or biochips are prominent among these new technologies. The use of microarrays for gene expression profiling, genotyping, detection of mutations and gene discovery are leading to remarkable insights into the function of thousands of genes previously known only by their sequence. GENE EXPRESSION : Each cell of the body (with few exceptions) contains a full set of chromosomes and identical genes. However, only a fraction of these genes are “turned on” at any given time for a given cell type. The subset of genes that are ‘expressed’ confer unique properties to each cell type. “Gene expression” is the term used to describe the transcription of information contained within DNA into mRNA molecules that are then translated into proteins that are involved in the critical functions of the cell. 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 acts as both an “on/off” switch to regulate 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. If we study the nature and amount of mRNA produced by a cell, we can learn which genes are expressed and gain insights into how the cell responds to its changing needs. DEVELOPMENT OF MICROARRAYS A DNA Microarray (which sometimes referred to as ‘gene chip’, ‘DNA chip’ or ‘gene array’) is a collection of microscopic DNA spots, commonly representing single genes arrayed on a solid surface (either a glass slide or a silicon chip) by covalent attachment. The DNA spots may be either oligonucleotides (oligonucleotide microarrays) or PCR products representing individual genes (cDNA* microarrays) (*cDNA = complementary DNA (cDNA) , the DNA synthesized from a mature mRNA template in a reaction catalyzed by the enzyme reverse transcriptase). The affixed DNA segments are known as ‘probes’. Thousands of such probes can be placed in known locations in a single microarray. The probe is complementary in sequence to the fragment of DNA or RNA to be detected (‘target’), so that it hybridizes selectively by Watson-Crick Base pairing to the correct (complementary) fragment of DNA or RNA if present in the test sample. Instead of detecting and studying one gene at a time, the DNA microarrays miniaturize the DNA Probe detecting method to allow thousands of specific DNA/RNA sequences to be detected simultaneously on a small chip of only 1-2 cm2. 1

XI ANNUAL CONFERENCE, IAMM AP CHAPTER : FEBRYUARY 2008, MEDICITI, HYDERABAD DNA microarrays can be used to detect DNA ( as in comparative genomic hybridization) . They are also useful for detection of mRNA (usually as cDNA, after reverse transcription) that may be translated to proteins. By using a microarray, in a single experiment it is possible to determine the expression levels of thousands of genes within a cell by measuring the amount of mRNA bound to each site on the array. This process is referred to as “expression profiling”. The use of microarrays for expression profiling was first discussed in 1987 and the arrayed DNAs were used to identify the genes whose expression is modulated by interferons. The earlier arrays were made by spotting cDNAs onto a filter paper with a pin-spotting device. The miniaturized ‘microarrays’ were available since 1996. The development of microarrays has been fuelled by the application of robotic technology to routine molecular biology. The classical Southern and Northern blotting techniques have provided the technological basis for microarray hybridization with fluorescently labeled cDNA. The idea of depositing multiple DNA spots representing different genes onto a solid surface was inspired from the work of Blattner et al (1993), who investigated the Escherichia coli gene expression on membranes (“macroarrays”). The recent application of robotics to achieve high spotting densities of DNA on glass slide was innovative and facilitates the construction of microarrays containing up to 50,000 genes on a single slide. FABRICATION OF MICROARRAYS : Two main methods are used to manufacture the microarrays – ‘spotted microarrays’ and ‘photolithographic technique’. The technology for producing spotted microarrays is widely accessible. In spotted microarrays, the probes are synthesized prior to deposition on the array surface and then ‘spotted’ onto a glass or silica chip. This is achieved by using an array of fine pins controlled by a robotic arm that is dipped into wells containing DNA probes and then depositing each probe at a designated location (to see the video demonstration, visit : http://en.wikipedia.org/wiki/Image:Microarray_printing.ogg ). The resulting ‘grid’ of probes is ready to receive the complementary cDNA or RNA targets. The second technique is Photolithographic synthesis , using the advances in the semiconductor technology. An array of oligonucleotide or peptide nucleic acid (PNA) probes is synthesized either in situ (on-chip) or by conventional synthesis followed by on-chip immobilization. This is limited to a small number of manufacturers like Agilens and Affymtrix. This method is costly but allows a very high spot density and consistency between ‘arrays’. “GENE HUNTERS” : THE APPLICATIONS OF MICROARRAYS The major applications of microarrays fall into three groups : 1. Gene expression profiling 2. Genotyping 3. DNA sequencing 2

XI ANNUAL CONFERENCE, IAMM AP CHAPTER : FEBRYUARY 2008, MEDICITI, HYDERABAD 1. GENE EXPRESSION PROFILING : The RNA is extracted from a complex sample such as body tissues or fluids or bacterial isolates is applied to the microarray. The result reveals the level of expression of thousands of genes in that complex sample. This is useful to study the effects of certain treatments , diseases or developmental stages on gene expression. Also it is useful to compare the gene expression in diseased and normal cells. The expression levels in two samples can be compared by dual fluorescent labeling. Simultaneous hybridization of two cDNA populations labeled with two different fluorescent dyes, Cy3 and Cy5 allows accurate assessment of relative levels of gene expression between the two different samples. The following diagram illustrates the procedure :

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XI ANNUAL CONFERENCE, IAMM AP CHAPTER : FEBRYUARY 2008, MEDICITI, HYDERABAD Description : the RNA extracted from the samples 1 and 2 ( or diseased and normal cells), cDNAs generated and labeled with red or green fluorescent dyes. The dye labeled samples are mixed and hybridized to the microarray. The RNA(cDNA) from each sample hybridizes to each spot on the microarray, in proportion to the level of expression of that gene in the sample. After hybridization, the red and green fluorescent signal from each sport is determined. The ratio of red to green reflects the relative expression of each gene in the two samples. This expression profiling has also an important role in pharmacogenomics . The goal of pharmacogenomics is to find correlations between therapeutic responses to drugs and the genetic profiles of patients. With expression profiling , it is possible to find out why some drugs work better in some patients than in others and why some drugs may even be highly toxic to certain patients. For example, if we want to study the effects of a potential new drug on liver, obtain two samples of liver cells and apply the drug to one sample. Then from each sample, the mRNA molecules are collected , cDNAs are synthesized and labeled with different fluorescent labels. By hybridization to the microarrays and analyzing the expression profiles, it is possible to determine whether any genes respond strongly to the drug to reflect liver damage. 2. Genotyping : Genomic DNA, extracted from an individual’s blood or saliva is amplified by PCR and applied to the microarray. The genotype for thousands of genetic markers can be determined which has considerable potential in risk assessment. 3. DNA Sequencing : The DNA extracted from an individual’s blood is amplified and applied to specific “re-sequencing” microarrays. Thousands of base pairs of the DNA can be screened for mutations in specific genes. Single nucleotide polymorphisms (SNP) among alleles can also be identified (SNP Detection arrays or SNP Chips). Such arrays are useful for detecting illness-linked gene variants and predict the person’s likelihood of developing Alzheimer’s disease, diabetes, cancer etc. such ‘at risk’ individuals can receive close monitoring, preventive care and early intervention. ROLE IN MICROBIOLOGY AND INFECTIOUS DISEASES Microarrays that detect gene sequences of Mycobactrium tuberculosis, HIV and other pathogens have been developed with the aim of providing a diagnostic tool that detects expression of antibiotic resistance genes or specific viral subtypes. These tests can be undertaken rapidly (in less than 24hrs) without the need for bacterial or viral cultures. This can be useful for earlier and targeted treatment based on antibiotic or antiviral sensitivities and will be particularly valuable for organisms such as M.tuberculosis and HIV for which sensitivity profiles can presently be determined only after lengthy analysis by other methods. Many international pharmaceutical and biotechnology companies are successfully using bacterial microarrays to drive programmes of novel drug development. Microarrays have also been used to perform high quality experiments to study the microbial environmental responses and global gene expression. The first global transcriptional profile was obtained for Saccharomyces cervisiae in 1997. By 1999, the microarrays for the genome of M.tuberculosis and E.coli were developed. Yeast has been the microorganism of choice for many 4

XI ANNUAL CONFERENCE, IAMM AP CHAPTER : FEBRYUARY 2008, MEDICITI, HYDERABAD research groups to analyse cell-cycle associated gene expression and the effects of various environmental changes, such as osmotic shock, temperature shock, presence of DNA-damaging agents and growth in minimal or nutritionally rich media. The whole-genome expression profiling revealed the complexity of the cellular response to major changes in metabolism as exemplified by the work on yeast diauxic shift by DeRisi et al (1997). In their work, the expression levels of 1840 genes (30% of a total of 6116 genes tested) were found to be affected by the transition from anaerobic to aerobic growth. Using expression profiling, it is possible to identify the cascades of gene expression during different phases of cell cycle and during sporulation. Microarrays are also helpful for genomotyping and strain comparison. Oligonucleotide-chip-based mutation analysis promises to be extremely powerful diagnostic tool for the identification of mutations in pathogens, such as mutations in rpoB gene (in a 75bp region) which can cause rifampicin resistance in M.tuberculosis. Mammalian gene microarrays have recently been used to study the host-pathogen interactions from the viewpoint of the host, by identifying gene expression patterns induced by the presence of a pathogen.

CONCLUSION : FROM MICROSCOPE TO THE MICROARRAYS Like the invention of the microscope a few centuries ago, DNA arrays hold promise of transforming biomedical sciences by providing new vistas of complex biological systems. At the most basic level, DNA arrays provide a snapshot of all of the genes expressed in a cell at a given time. Since gene expression is the fundamental link between genotype and phenotype, DNA arrays are bound to play a major role in our understanding of biological processes and systems ranging from gene regulation, to development, to evolution, and to disease from simple to complex. For instance, DNA arrays should play a role in helping us to understand such difficult problems as how each of us develops from a single cell into a gigantic supercomputer of roughly 1015 cells, and why some cells proliferate in an uncontrolled manner to cause cancer. One notable difference between modern DNA array technology and the seventeenth-century microscope, however, is in the output produced by these technologies. In both cases, it is an image. But unlike the image one sees through a microscope, an array image is not interpretable by the human eye. Instead, each individual feature of the DNA array image must be measured and stored in a large spreadsheet of numbers with tens to tens-of-thousands of rows associated with gene probes, and as many columns or experimental conditions as the experimenter is willing to collect. As a side note, this may change in the future and one could envision simple diagnostic arrays that can be read directly by a physician.

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XI ANNUAL CONFERENCE, IAMM AP CHAPTER : FEBRYUARY 2008, MEDICITI, HYDERABAD REFERENCES AND RESOURCES : Chuang, S. E., Daniels, D. L. & Blattner, F. R. (1993). Global regulation of gene expression in Escherichia coli. J Bacteriol 175, 2026-2036. Cummings, C. A. & Relman, D. A. (2000). Using DNA microarrays to study host–microbe interactions. Emerg Infect Dis 6, 513-525. DeRisi, J., Penland, L., Brown, P. O., Bittner, M. L., Meltzer, P. S., Ray, M., Chen, Y., Su, Y. A. & Trent, J. M. (1996). Use of a cDNA microarray to analyse gene expression patterns in human cancer. Nature Genet 14, 457-460. DeRisi, J. L., Iyer, V. R. & Brown, P. O. (1997). Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278, 680-686. Lucchini, S., Thompson, A. & Hinton, J. C. D. (2001). Microarrays for Microbiologists. Microbiology 147, 1403-1414. Hinton, J. C. (1997). The Escherichia coli genome sequence: the end of an era or the start of the FUN? Mol Microbiol 26, 417-422. Manger, I. D. & Relman, D. A. (2000). How the host ‘sees’ pathogens: global gene expression responses to infection. Curr Opin Immunol 12, 215-218. Schena, M., Shalon, D., Davis, R. W. & Brown, P. O. (1995). Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467-470. Stephen H Friend, Roland B Stoughton (2002). The Magic of Microarrays. Scientific American 2002(Feb), 34-41. Timothy J Aitman (2001). Science, Medicine and the Future : DNA Microarrays in Medical Practice. BMJ 2001;323;611-615.

Links to Web: http://microarray.wordpress.com/2007/11/06/microarray-based-clinical-diagnostic-tests/ http://microarray.wordpress.com/2007/11/04/new-micrrray-chips-technology-will-replace-pcr-andspeed-up-the-hts/ http://www.affymetrix.com/index.affx http://en.wikipedia.org/wiki/DNA_microarray http://www.healthtech.com/glossaries/content/microarrays.htm http://www.bio.davidson.edu/Courses/genomics/chip/chip.html

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