Emerging technologies
Recent advances in microarrays

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An overview is presented for the current DNA-based microarray market, including applications for microarrays in areas such as gene expression, single-nucleotide polymorphism, strain differentiation, de novo DNA synthesis, aptamers and advances in ‘in situ’ synthesis technology. The development of new detection methods, simplified methodologies and broad application to molecular diagnostics are rapidly migrating microarray technologies into the arena of diagnostics and personalized medicine. Comparisons of microarray technologies from various manufacturers are presented.

Section editors:

Steve Gullans – RxGen, Inc., New Haven, CT, USA

Robert Zivin – Johnson and Johnson, New Brunswick, NJ, USA

Introduction

Over the past 5 years, new methods of manufacturing microarrays have appeared in the market place, which are superceding more traditional spotted array formats. The propagation of novel in situ synthesis technologies for DNA synthesis on chips allows distinct microarray designs to be manufactured within days rather than weeks or months. In situ synthesis also results in a highly economical production of many distinct oligonucleotides, avoiding the need to make bulk purchases of sets of oligonucleotides for spotting. In addition to being fabricated by the manufacturer, arrays can now be manufactured by customers at their own facilities using commercially available DNA chip synthesizers.

Applying elements of manufacturing technology from the semiconductor industry has significantly improved microarray manufacturing. Recent developments in semiconductor materials and electrochemical DNA synthesis are now bringing semiconductor devices to the forefront of microarray technology.

Biochips are composed of a solid support to which the DNA molecules are attached. For spotted oligonucleotide arrays, the chip surface contains activated chemical moieties to couple the oligo to the surface. By contrast, in situ synthesis systems use a membrane or a porous reaction layer over the chip surface to support DNA synthesis. Biological materials are bound to these activated materials using a variety of chemistries [1, 2, 3, 4, 5, 6, 7]. Most crucially, these linker and coating chemistries maintain this attachment, even after the chemical removal of DNA-protecting groups or hybridization [7].

The means of attachment to the ‘substrate’ as well as the material/chemistries used vary. In some cases, it is a direct attachment to some activated groups on the surface of the material used (e.g. epoxy-modified glass slides) or it might simply be a hydrophobic interaction. In other cases ‘biological glue’, such as biotin or other hapten ligands, might be used to tether molecules robustly on the surface. The utilization of biotin allows the use of streptavidin molecules because biotin has an extremely high affinity for streptavidin (Kd  10−15) and streptavidin contains multiple biotin-binding sites.

There are several types of ‘biochips’. The most common are DNA chips, where snippets of DNA ranging from 25 bases (oligonucleotide arrays) to hundreds of bases in length (cDNA arrays) are laid down or synthesized in situ on the chip. Several peptide and protein chips are available on the marketplace [8, 9, 10, 11, 12] and several systems for performing peptide synthesis in situ have been developed [13, 14]. For larger proteins, in situ synthesis is not an option and spotting remains the primary method for making arrays. Additionally, several other types of biochips exist including glycobiology chips as well as ‘organic chemistry-based combinatorial chips’ [15, 16, 17, 18, 19].

DNA chips have been prepared by numerous means but they are broken down to two categories: spotted arrays and synthesized arrays. In situ synthesized arrays are generated by spatially controlled patterning techniques including photolithography, ink-jet synthesis and electrochemical patterning on an electrode array [7, 20].

Detection and labeling methodologies for DNA chips vary widely. Traditionally, fluorescence detection methods abound as labels are readily available and laser-based scanners are sold by several companies. Fluorescent labels are available as modified bases or are conjugated chemically after nucleic acid amplification. The scanners might be generic, usually designed to scan a 1 in. × 3 in. microscope slide format. By contrast, some are restricted to a particular company's chip dimensions. Although this might offer some performance advantages because of the scanner being tuned to the system, this tends to bind the user to a particular experimental approach. Another detection method is luminescence, with the advantage of a very low intrinsic background. Utilizing enzyme-based luminescence provides some enhancement over fluorescent-based imaging systems [21]. A third detection method is redox enzyme amplification electrochemical detection [20, 22, 23, 24, 25]. Advantages of electrochemical detection include the elimination of the image-analysis step required by all fluorescent or luminescent imaging systems because electrochemical measurement produces a value of current per electrode rather than an image. Neither electrochemical nor luminescent imaging systems suffer from the known problems associated with high-resolution fluorescent imaging, namely ozone sensitivity and photobleaching/quenching effects.

Chip density and oligonucleotide length vary between platforms, as shown in Table 1. The choice of platform depends on user requirements for flexibility, cost and capability. By contrast, proteins and carbohydrate chips tend to have lower densities because the assay tends to be harder to optimize to one set of binding conditions. DNA chips have a distinct advantage in this regard because it is possible to optimize the probe design on the chip to result in optimal performance at a single set of reaction conditions.

Section snippets

Gene expression analysis/transcriptional profiling

This is the classical assay that was developed using DNA chips. In this case, differential gene expression information is obtained from pairs of RNA samples. This is usually as a function of cellular responses to the action of specific signals, disease states or drugs. Typically, two RNA samples are prepped and tagged with different fluorescent dyes [26]. The use of a standard then allows the detection of silenced/suppressed genes or activated genes. In general, a fair amount of variability

New detection methods

Optical detection methods for microarrays, such as fluorescence measurement with laser scanners have been employed successfully for over a decade. However, optical imaging and image analysis add complexity and can be a significant source of noise depending on the calibration status of the instrument.

Electrochemical methods available on the market (for molecular diagnostics) rely on ‘single pass’ detection methods and in some cases they oxidize (destroy) the material being tested.

Conclusion: summary comparison of microarrays

We have compared the current DNA microarrays that are available in the market place in terms of their manufacturability and easy use. We have gone into some depth to discuss the oligomer lengths and synthesis along with the capabilities of each chip system. The in situ synthesis arrays provide a new level of customizability not found in earlier systems and allow the development of a suite of new applications for microarrays. In addition, the faster turn-around time for custom synthesis

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