New Methods for Gene Expression Profiling

In the last 5 years of the twentieth century, and subsequently, there has been rapid growth in the development of alternative techniques for measuring gene expression on a large scale. There are two principal drivers behind the growth of diverse measurement techniques. The first is simply the commendable attempt to provide technologies that are more reliable, reproducible, precise, and cost-effective. The goal, after all, is to be able to make the measurement of a set of gene profiles no more expensive than a simple serum chemistry drawn routinely during a doctor's visit. It should be robust and affordable enough to be deployable widely for purposes of microbial genome detection for public health surveys and for routine screenings for a variety of diseases during clinical care and well-care checkups. Unfortunately, the world of commercial intrigue and murkiness around intellectual property has been the second important driving force in developing these technologies. Affymetrix, for example, has a patent that, under some interpretations, might include a range of microarray gene expression measurement technologies on a two-dimensional surface. Consequently, much of the development of alternative technologies has been focused on avoiding the potential condflict with this intellectual property (see figure 7.1).

Who's Suing Whom

Plaintiff -^ Defendant

Litigation as of November 30, 2000 Figure 7.1: Lawsuits in the microarray field. With the number of lawsuits already in this area, it should be no surprise that companies in this industry are striving to determine new and unique intellectual property. (From Gibbs et al [77].)

We review here some of the major and upcoming technologies. Perhaps the best way to think of these technologies is within the following framework. Some of them use full-length cDNA probes, others use oligonucleotide probes. Independently of what kind of probe they use, the technologies may use a solid two-dimensional surface, a three-dimensional matrix, or optically coded beads.

We are hardly going to be able to even enumerate all the emerging technologies for expression measurement. For the latest up-to-date list, the reader is directed to http://www.gene-chips.com/. Also, for an excellent review of the scientific underpinnings of many of these technologies, we refer the reader to a review by Southern et al. [166] which provides a survey of the physicochemical basis of molecular interactions of hybridization on microarray surfaces. We would also impress upon the reader that most of the technologies described either have only begun early production lines or are still under development. Consider that it has taken well over 3 years, and counting, for the mainstays of microarray expression measurement, oligonucleotide microarrays, and robotically spotted microarrays to achieve minimal levels of accuracy and reproducibility. Then, as interesting and appropriate as some of the following methodologies may appear, they bear close scrutiny and testing prior to any large scale attempts for use in scientific experimentation.

Additionally, each of the technologies described is, of necessity, undergoing iterative processes of improved quality, higher density, and lower cost. This makes it all the more challenging to decide which microarray platform to adopt. This is very much analogous to choosing a home video gaming system: Nintendo, Sony, Microsoft, each has its own game console. As each manufacturer rolls out its next-generation platform at different times, consumers have to agonize over whether it is better to buy the platform that best suits their needs today or the one that will do twice as much in 4

months. As the refinement of these platforms is an iterative, continual, and competitive process, there is no stable or consistent "correct" response to the consumers' dilemma.

The video gaming console analogy is also relevant to the commoditization of microarray platforms. In the 1980s, real-time three-dimensional rendering was the province of highly specialized, expensive computer systems available in only a few laboratories. The cost of the hardware and staff far exceeded the cost of the system software. Now, with commodified video consoles, over the short life of a consumer system, the major cost is the acquisition of software and game modules. Similarly, the current microarray systems are labor-intensive and the hardware (including the microarrays themselves) and staff drive the high costs of expression profiling laboratories. As a result of the competition and improvements in manufacturing technology, the costs of hardware and attendant staff will diminish and likely become dominated by the costs of tissue acquisition, banking, and annotation.

With these issues in mind, we review some of the available and upcoming platforms. 7.1.1 Electronic positioning of molecules: Nanogen

Most biological molecules have a natural positive or negative charge. When these molecules are exposed to an electric field, those with a positive charge move to an area with a negative charge, and vice versa. Novel microarray technologies such as those of Nanogen (San Diego, CA) use this property to maneuver molecules to specific test sites on an electronic microchip. By carefully controlling which element of a grid of electrodes is charged, this technology can direct many test molecules to specific areas of the chip. Because of the more active nature of this technique, this could potentially deliver molecular binding on the microarray several orders of magnitude faster than passive hybridization methods.

Regarding gene expression measurement, a cDNA molecule that is negatively charged moves in an electric field to an area of net positive charge. The sample DNA is significantly concentrated over time in the area of positive charge. DNA that does not have the right mass/charge ratio is repelled from the area of the electrode under closely controlled electronic conditions (see figure 7.2). The current trade-off (pun intended) for all this configurability is that the density of features of these microarrays is at least one order of magnitude less than the current density offered by companies such as Affymetrix. However, the density of this technology is rapidly growing.

Figure 7.2: The Nanogen microelectronic array.

This system may be best suited for laboratories that are interested in a medium-throughput platform that will allow several genomic applications such as gene profiling, proteomics, and genotyping.

Figure 7.2: The Nanogen microelectronic array.

This system may be best suited for laboratories that are interested in a medium-throughput platform that will allow several genomic applications such as gene profiling, proteomics, and genotyping.

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