DNA Microarrays: An Overview of Technologies and Applications to Toxicology
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- Abstract
- Table of Contents
- Figures
- Literature Cited
Abstract
DNA microarrays or chips can be used to simultaneously monitor the expression levels and/or genotypes of thousands of genes. The application of these techniques heralds a new era in toxicology research, where genotypes and toxicant?induced expression signatures may be used to monitor cellular responses to different doses, to classify toxins on the basis of their mechanisms of action, to monitor exposures, and to predict individual variability in toxicant sensitivity. This unit reviews the current state of microarray technologies and discusses potential applications in toxicology, with emphasis on the strengths and limitations of the technologies.
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- Aims and Objectives
- Development of DNA “Chips”: A Quantum Leap in Molecular Detection Technology
- Analysis of Gene Expression Patterns Using Nucleic Acid Arrays
- Application of DNA Chip Technology to Detect DNA Sequence Polymorphisms (SNPs)
- Computational Biology
- Literature Cited
- Figures
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Figure 1.4.1 Expression analysis using cDNA microarrays. To produce cDNA microarrays, individual cDNA clones are amplified by PCR and arrayed in a multiwell format with or without purification. The upper left panel shows samples being arrayed with a piezoceramic fluid dispensing system (Engineering Arts). The multiwell plates with cDNA clones are then placed on a robotic printer such as the Gene Machines system shown in the second panel. The software that drives the robot is used to assign each clone to a specific address on the surface of a polylysine‐coated or chemically derivatized microscope slide. Spotting of individual clones is accomplished using a series of pins or quills that are dipped into each well to take up a sufficient volume of the cDNA solution to spot or print each clone onto all of the slides. Printing is accomplished by rapid displacement of the robot head and the microscope stage using precision micromanipulation technologies. After each round of printing, the pins are washed and the process repeated until all clones have been printed on each slide to produce a microarray. View Image -
Figure 1.4.2 Expression analysis using oligonucleotide arrays. The figure shows expression patterns for (A ) normal human squamous epithelial cells and (B ) chronically exposed cells (M.T. Barrett, K.Y. Yueng, P.L. Blount, R.C. Sullivan, H. Zarbl, J. Delrow, and B.J. Reid, unpub. observ.) using oligonucleotide GeneChip Expression arrays produced by light‐directed chemical synthesis (Affymetrix). Each array comprises tens of thousands of probes that interrogate the expression levels of ∼1700 genes. Messenger RNA extracted from each cell type is reverse transcribed into cDNA and is subjected to linear amplification by in vitro transcription in the presence of biotinylated oligonucleotides. The amplified cRNA targets from each cell are hybridized to a separate microarray. The amount of each target sequence hybridized to a probe is quantified by binding of strepavidin phycoerythrin, excitation with laser light, and collection of fluorescence intensity data using a Hewlett‐Packard scanner. View Image -
Figure 1.4.3 Detection of single nucleotide polymorphisms (SNPs) using oligonucleotide arrays. This is an example of the data obtained for individuals that are wild‐type homozygous (WT), heterozygous (Het), or mutant homozygous (Mut) for a specific allele of the P450 cyp 2D16 gene. Experiments were performed using the GeneChip P450 oligonucleotide arrays produced by light‐directed chemical synthesis (Affymetrix). For each polymorphic site, there is a series of five columns of probes that interrogate the polymorphic base and 2 bases on either side. Columns from left to right interrogate sequences at position ‐2 to +2 relative to the site interrogated and alternate from left to right between wild‐type (W) and mutant (M) probes. The columns are each comprised of four probes (4 L ) that collectively detect all 4 possible nucleotides at each position within the 5‐bp sequence. The presence of wild‐type and mutant alleles is determined by the pattern of hybridization with probes that detect wild‐type and/or mutant targets. For SNP detection, genomic DNA is extracted and a region of several hundred base pairs flanking each polymorphic site is amplified by multiplexed PCR. After verification that each region of interest was amplified, the PCR products are fragmented to a size optimal for hybridization and 3′‐end labeled using fluoresceinated deoxyribonucleosides and terminal transferase enzyme. Following hybridization and scanning, the data are analyzed by appropriate software and genotypes are assigned to each sample. View Image
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| Key References | |
| Supplement to volume 21 of Nature Genet. (1999) The Chipping Forecast. | |
| This supplement consists of fourteen comprehensive articles written by the leaders in the field of microarray technologies. The reviews provide detailed descriptions of all aspects of array technologies, including the chemistry and interactions occurring in the array surface, reviews of different technologies and applications, how to build and run an array facility, detailed protocols for hybridization, specific research applications, and overviews of data management and statistical analysis, including cluster analysis and data exploration. | |
| Internet Resources | |
| http://www.ncbi.nlm.nih.gov/Genbank/index.html | |
| A public database of all DNA sequence entries. | |
| http://www.ncbi.nlm.nih.gov/Entrez/Genome/org.html | |
| A public database of all completed genomic sequences. | |
| http://www.affymetrix.com/technology | |
| A commercial Web site provided by Affymetrix of Santa Clara, California. The site provides detailed descriptions of chip production using photolithography and includes video of the process. Complete description of products, publications, etc. are also provided at this site. | |
| http://cmgm.stanford.edu/pbrown/mguide/index.htm | |
| Web page provided by the laboratory of Patrick O. Brown at Stanford University. Provides detailed information on building a robotic arrayer and using cDNA arrays for expression analysis. |
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