for study of
Genetic Mutations and Diseases,
Allelic Variations, and Genetic Fingerprinting.
John D. FurberMaster of Science, Biological Sciences, University of California, Irvine.
Bachelor of Arts, Physics and Mathematics, University of California, Santa Cruz.[ John D. Furber home page ]
Note to Readers:Most of the following review was written in late 1999 and early 2000, when microarrays were not yet in widespread use. Since that time, the use of microarrays by biologists has greatly increased, and the number of companies producing or supplying arrays, readers, and analysis software has increased.
The Human GenomeThere are roughly between 30,000 and 120,000 genes in the human genome. As of early year 2000, most of them are of still unknown function. This is changing rapidly, as new tools are being developed which are vastly accelerating our ability to find genes and study their function. (Link to Genomics Research)
Differential Gene ExpressionAs humans grow, age, and live their lives, various subpopulations of cells in their body can be distinguished by form, behavior, and by turning on and off the expression of various subsets of the genome which nearly all cells contain. In some cases, the on and off expression cycles last for seconds or hours to respond to environmental or metabolic changes. Other genes are turned on or off for life when cells commit themselves to various fates, such as becoming a neuron or a muscle cell. Studies of gene expression patterns are of enormous importance to Biomedical researchers seeking to understand the functioning of normal and diseased cells, as well as the aging process. We can estimate the expression level of a particular gene in a cell by measuring the number of copies of the mRNA or protein molecules encoded in that gene. (Link to Developmental Biology Research)
Dot BlotsEarly arrays employed rather large spots (on the order of a centimeter), which limited the number of array elements which would fit in a given area. [Costanzi, Spoerel] They were named "Dot Blots" for the round shape of the spot which forms when a drop of target solution soaks into the nitrocellulose paper or nylon filter substate. At that time, it was common for Developmental Biologists to dot RNA or cDNA from various tissue types, or developmental stages, or species as array elements, and then to probe with DNA cloned from their gene of interest. Researchers at that time were only studying one or a few genes per experiment, and this procedure provided them with useful information about where and when their gene was expressed.
DNA microarraysDNA microarrays can achieve higher densities than nitrocellulose dot blots because very small quantities of DNA are bound to glass substrates, and they don't spread around as widely. The rigidity of the substrate and new, high-resolution laser scanning technology, combined with bright, instant-reading fluorescent tags, allows signals to be distinguished and associated with very small dots, very close together. A one-inch square can easily hold a 100 X 100 array, which is 10,000 distinguishable spots [Iyer].
Two somewhat different microarray technologies are competing in the market:
Expression Microarrays are DNA microarrays which can analyze the expression levels of many thousands of genes simultaneously. High-density expression microarrays now make it possible to analyze the expression of all genes simultaneously. The arrays are made by binding precisely measured quantities of unlabeled, EST molecules to a glass slide. In use they are rinsed with labeled, single-stranded mRNA or cDNA mixtures from the cells of interest. Complementary DNA or RNA molecules hybridize to each other, attaching the fluorescent molecules to particular spots on the array. By looking at which grid points light up, we see which genes were producing mRNA in the cells. Furthermore, the brightness of the points can be precisely measured with instruments to yield a quantitative measure of the number of copies of each mRNA species which stuck to the array. Statistical analysis then relates the number of bound mRNA molecules to the likely number present in each cell at the moment of harvesting.
- cDNA microarrays, developed by Stanford University and Incyte Genomics.
- oligonucleotide microarrays, developed by Affymetrics.
Diagnostic Microarrays: This same microarray technology also has the capability of examining thousands of different genes from a single small tissue sample to determine which are normal and which contain mutations. Diagnostic microarrays often contain synthesized DNA sequences on the grid points of the glass slide. They can then be hybridized with genomic DNA from the individual patient to determine which allelic sequences it binds to, and therefore which alleles are present in the patient's genome. For a catalog of human genetic diseases, see the OMIM Database.
Genetic and Gene Expression Research with Microarrays
Microarray Manufacturers in the San Francisco Bay Area
- Affymetrix, Inc. in Santa Clara makes DNA microarrays on glass wafers, which it calls "GeneChip(R) DNA probe array technology." Affymetrix synthesizes single stranded DNA oligonucleotides of about 20-25 bases in length right on the array chip, using masking techniques adapted from the silicon integrated circuit industry. In their vocabulary, the oligonucleotides on the chip are refered to as "probes," and the DNA rinsed over the chip is called the "target." Affymetrix has teamed up with Roche Molecular Systems, Inc. to produce clinical diagnostic kits using GeneChip probe arrays. Some or all of these kits are likely to be for allele assays of individual genomes, rather than for gene expression analysis. [Schmidt] The current GeneChip system includes a scanner manufactured for Affymetrix by Hewlett Packard, a fluidics station for sample handling, and a computer workstation and associated software for data analysis. Affymetrix is now distributing its GeneChips thorugh Amersham Pharmacia Biotech.
- HySeq, Inc. in Sunnyvale has developed a microarray DNA chip system, which it calles the HyChipTM Sequencing Chip, in collaboration with Perkin-Elmer Biosystems, for diagnostic and research applications. "The HyChipTMsystem utilizes arrays of a complete set of DNA probes in conjunction with a target-specific cocktail of labeled probes to identify single nucleotide differences between a reference and test sample."
- Incyte Microarray Systems (formerly Synteni, Inc.) in Fremont, California has an exclusive license to Stanford's DNA on glass microarray technology, and is selling its GEMTM (Gene Expression Microarray) chips commercially. In the Stanford/Synteni system, expressed cDNA sequences 500-5000 bases long are amplified in individual tubes by PCR. Robotic quill pens deposit a tiny drop of DNA solution onto a poly-lysine coated glass slide, one drop for each gene at precisely the correct array location. Synteni, Inc. was acquired by Incyte Genomics, Inc. (formerly Incyte Pharmaceuticals, Inc.) in January 1998. Numerous U.S. and foreign patent applications are currently pending, covering many broad aspects of this technology. GEM technology can currently fit 10,000 unique genes on a single array. Incyte Microarray Systems now provides pre-fabricated and custom microarrays and services for applications in human, animal, microbial, and plant genomics. They are now arraying Incyte's cDNA clone libraries onto glass wafers using Synteni technology. Incyte has combined its in-house bioinformatics expertise and genomic databases to develop several new prefabricated microarrays. Each contains up to 10,000 sequence-verified, Incyte-proprietary clones from their LifeSeq, ZooSeq, and PathoSeq databases and their GeneAlbum reagent set. They also offers several microarrays based on public-domain genes. The UniGEM Series 1-4 covers 40,000 human genes from NCBI's UniGene database and the Mouse Series 1-2 under development will contain 18,000 putative mouse genes.
- Stanford University in Palo Alto has been instrumental in developing and using expression microarrays for biomedical research. A Stanford web site shows details of how to build and use the technology.
Reading List and References
- C Costanzi, D Gillespie. Fast Blots: Immobilization of DNA and RNA from Cells. Methods in Enzymology, Vol 152 Guide to Molecular Cloning Techniques. (Academic Press, 1987).
- VR Iyer, et.al. The Transcriptional Program in the Response of Human Fibroblasts to Serum. Science. 283, 1 Jan 1999 pp. 83-87, (endnote #21).
- KF Schmidt. Just for You. New Scientist. 14 Nov 1998, pp. 32-36.
- NA Spoerel, FC Kafatos. Identification of Genomic Sequences Corresponding to cDNA Clones. Methods op. cit.
Contact information:Please send updates, corrections, and questions to:
John D. Furber
E-mail: johnfurber (at) aol (dot) com
[ John D. Furber home page ]
© 2000 - 2005 by John D. Furber. All rights Reserved.