Chips for the Common Man

Just when you might be thinking that the DNA chip revolution is passing you by, a number of companies are coming out with products aimed at serving the needs of the "average" researcher. The day may be coming when you don't need to use blow your entire supply budget (or sell your first born) to run an experiment on DNA chips. This profile is the first of a series that will take a look at products and technologies that have appeal to those with modest research budgets, and will consider some the cost/benefit issues that arise when deciding among the various technologies available for expression monitoring. Future articles in this series will look at other aspects of the DNA microarray revolution—such as expanding the range of data collection through the use of multiple labels, and software solutions for handling the large data sets that microarrays generate.

What's New?
Array Away
The New Kid on the Block
The Genes
TSA Amplification
The Proof Is in the Pudding
More to Come

What's New? (Back to Top)
More companies are entering this already competitive field of DNA microarray technology every day, or so it seems. Two brand new entries into the fray are NEN Life Sciences (Boston, MA), which will be introducing a new chip array system, the MicroMax, this summer, and Axon Instruments (Foster City, CA), which has introduced its first product in its genomics product line, the GenePix 4000 Scanner. In addition, General Scanning (Watertown, MA) has added to its existing product line with the ScanArray 5000, a four-laser scanner that leads the field in flexibility, and GenPak (Stony Brook, NY) is launching both a manual and an automatic arraying system.

Array Away (Back to Top)
Researchers interested making use of this technology have a variety of options. They can make their own arrays, by using one of the several commercially available arrayers or by fashioning their own (plans for a microarrayer are posted on the web site of Pat Brown of Stanford University). Alternatively, they can purchase an array from a number of companies.

Arrays come in two varieties—macro and micro—which differ in the size of the spot, as well as the over-all dimension of the array. (Macro arrays typically have spots 300 microns or greater and are arrayed on membrane filters anywhere from 22 cm² to almost 100 cm², while microarrayed spots are 200 microns or less, and are getting smaller all the time.) Filter arrays have certain advantages in that the equipment required for their use is available in most modern molecular biology laboratories, and they can be used with isotope, which reduces the amount of probe required. However, they have certain disadvantages, to wit each sample must be analyzed with a separate filter, unlike microarrays, onto which multiple fluorescent probes can be applied simultaneously.

Filter arrays that contain Image clones (all expressed clones, though not all identified) can be purchased from Genome Systems (St. Louis) and Research Genetics (Huntsville, AL), while Clontech (Palo Alto, CA) and Sigma-Genosys (The Woodlands, TX) provide filters dedicated to particular research areas or developmental states, such as apoptosis, cancer, or cytokines.

Getting into the high-density arrays (the microarrays) will cost a bit more, but then again, you also get more information from these systems. (The actual cost per gene is pennies, if you care to think of it this way.) Affymetrix (Santa Clara, CA) produces GeneChip arrays for looking at the totality of expression from yeast and large portions of the human and mouse genome. They also make chips to analyze polymorphic regions of individual genes of clinical relevance, such as the cytochrome P450 gene, the HIV protease and reverse transcriptase genes, and p53 gene. The chips, as well as the workstations and imagers, are available through Amersham Pharmacia Biotech (Piscataway , NJ).

Researchers who don't want to get their hands wet can purchase the services of Incyte (through Genome Systems) or HySeq (Sunnyvale, CA). And finally, custom chips are an option offered by several of the chip giants—Affymetrix, Incyte—as well as the not-so-giant Genometrix (The Woodlands, TX), which produces low to medium density arrays relatively inexpensively.

The New Kid on the Block (Back to Top)
NEN Life Sciences is coming out with a DNA array system in the hopes of breaking down some of the barriers of expense and time that may be prohibitive to some who wish to make use of this technology. The Micromax Human cDNA Microarray System puts everything you need for expression profiling in a single package. At the center of the system is the microarray itself, a pre-spotted glass slide that contains 2,400 known human genes. Reagents for preparing cDNA probes are included, as well as NEN's patented tyramide signal amplification (TSA) system for amplification and detection of cDNA probes. In addition, the company maintains a web-based database for every gene on the array to assist in data interpretation. NEN even provides a scanning and image analysis service for those without an appropriate scanning device at hand.

The Genes (Back to Top)
On the first of what will be a series of DNA chips, some 2,400 known human genes derived from more than ten tissues are arrayed on a 1-inch by 3-inch glass slide (provided in duplicate). These cDNAs were created by AlphaGene (Woburn, MA), using its patented FLEX (full-length expressed gene) technology, and come from over 50 libraries. Only genes whose functional domain is known are included on this first chip, and representatives of the 21 classes of Prosite domains—receptors, growth factors, cytokines—are among the 2,400 genes. NEN maintains a listing of the genes as well as links to gene banks on its web site.

TSA Amplification (Back to Top)
A unique feature of the NEN Micromax system that by itself warrants a closer look is its TSA amplification system. Unlike other methods that amplify the target, in this system, a catalytic process is used to amplify the amount of signal. Biotin- or DNP-labeled probe is hybridized to the chip, followed by antibody to the label, conjugated with horseradish peroxidase. The addition of fluorescent-labeled tyramide (Cy3 or Cy5) leads to its deposition at the site of labeled cDNA probe. The system is quick (less than 10 minutes) and requires 10- to 50-fold less probe than the unamplified method (see figure). Company experiments have shown that with the TSA amplification system, RNA present in one to two copies per cell can be detected starting with only 200,000 cells.

The Proof Is in the Pudding (Back to Top)
Shown below is an example of an expression profile done with Micromax, comparing normal epithelial cells with a melanoma cell line (903 cells), with and without signal amplification. With TSE amplification, you clearly can see more sites of hybridization, as well as brighter signals throughout, using 50-fold less probe. Scientists at NEN have been able to validate the system by quantitating the expression level of genes known to be elevated in particular cell types. For example, in prostate cancer cells, the mRNA for PSA (prostate specific antigen), which is over-expressed in prostatic cancer, was found to be elevated 13-fold over normal prostate cells.

More to Come (Back to Top)
The Micromax System, priced at around $2,000, provides a simple, cost-effective (pennies per gene), and sensitive method for monitoring gene expression. And there are more chips on the way. In the near term, NEN plans to introduce a series of "focused" chips, which will contain arrays of families of genes.

For more information on the Micromax System, contact NEN Life Sciences, 549 Albany Street, Boston, MA 02118-2512

Next up in this series: Hazel Richards of GenPak Ltd. will step you through making an array using the GenPak GeneStation 3, an inexpensive workstation for hands-on arraying.

By Laura DeFrancesco