Hybridization Array Technologies

Macroarrays

The term macroarray has been applied to formats in which areas of probe localization, often called features , are large enough to be visualized without magnification. These have been manufactured on nylon or nitrocellulose membranes or as plastic strips with linear arrays of bound target. Because of the size of the features, the density of macroarrays is much lower than microarrays, ranging from a few dozen to hundreds or even thousands of probes. They are usually deposited onto the membrane by printing or dot blotting and then dried and stored for future use.

Nylon, plastic, and glass (silicon wafer) are standard supports to make macroarrays. Nylon suffers several drawbacks compared with silicon, which is a preferred matrix for hybridization arrays. Besides its porous nature, nylon displays high autofluorescence, limiting the sensitivity of fluorescence-based detection because of high background values. The latter also precludes the possibility to develop ratiometric (or two-color) assays. This approach uses two targets, each labeled with a different fluor. Most commonly, one is a constant reference control used in direct comparison with the unknown sample, with results expressed as a ratio of the two emitted fluorescent signals ( ).

Macroarrays are currently used for targeted applications. For example, some commercial arrays target all the known cytokine genes or those within specific signal transduction pathways that are activated during infectious and inflammatory processes. Other arrays, targeting the genes altered during oncogenesis, have been used in cancer research (e.g., Atlas Human Arrays, Clontech ; GeneFilters, Research Genetics). Other commercial applications, usually in kit format, are used to detect and type specific PCR products, such as in the cystic fibrosis mutation screen, human leukocyte antigen typing, or human papillomavirus (HPV) typing. Furthermore, nylon-based arrays have been used to detect bacterial colony–based gene expression ( ) and thyroid cancer detection ( ). The advantages of macroarrays include the ability to use standard hybridization equipment, simplified reading or scanning, and affordability.

Some groups have been successful in producing high-density nylon arrays on a custom basis for gene discovery purposes ( ; ; ; ; ; ). Even in high-density configurations, the size of the nylon arrays requires a relatively large volume of hybridization fluid, a practical limitation with respect to generating a probe from small amounts of diagnostic material. Because microarrays, rather than macroarrays, represent the high-density, high-throughput platform of choice, the bulk of this chapter focuses on applications of microarrays.

Microarrays

Assay miniaturization saves time while cutting costs in biomedical diagnostic applications and research. Working with smaller volumes reduces reagent consumption, increases sample concentration, and improves reaction kinetics. These improvements allow the investigator to determine hundreds or thousands of results in the time formerly required for a single experiment. Microarrays are now available from several commercial suppliers, along with reading and fabrication equipment for custom manufacture of dedicated arrays for specific research or diagnostic applications.

Microarray Substrates

A major distinction between microarrays and macroarrays consists of the choice of a nonporous solid support. By preventing diffusion of the target nucleic acids, nonporous surfaces (plastic, glass, or silicon substrates) allow faster hybridization kinetics and easier washing steps. To allow spatial discrimination of numerous reactions performed simultaneously, the molecular probes are bound to a solid surface in defined arrays. Deposition of probes on a solid substrate is also more amenable to automation and enables higher array densities with optimal image definition. These features apply to any type of microarray, from synthetic oligonucleotides to cloned cDNAs or PCR products ( ). Most microarrays are now developed on glass. As opposed to plastic derivatives, glass transparency and lack of autofluorescence allows low-background, fluorescence-based detection. However, as materials science becomes more involved in array technologies, alternative substrates, including coated glass and plastic substrates, may become available.

Microarray Fabrication

3′-End functionalization (i.e., chemical modification) of nucleic acids—oligonucleotides, PCR products, cDNAs, or peptide nucleic acid oligomers—is required for covalent immobilization on either glass or polypropylene surfaces ( ; ). For example, treatment of glass slides with silane allows amino-covered glass to bind amino-linked probes, using bifunctional molecules such as a dialdehyde or a diisothiocyanate ( ; ). Alternatively, glass coating with a polycation (e.g., polylysine) allows direct charge-coupled binding of polyanionic DNA probes ( ); an ultraviolet photo-cross-linking step adds covalent bonds to the ionic interaction. Covalent binding is essential to permit stringent washes and therefore accurate discrimination of hybridized species. Several protocols have been published to address surface activation ( ; ; ; ). An interesting alternative consists of deposition of small patches of activated polyacrylamide to which presynthesized oligonucleotides are attached by microinjection ( ; ; ).

The packing density of probes attached to solid surfaces influences greatly the performance of hybridization matrices. Poor coupling efficiency, which is often encountered on glass matrices, can result in low probe density and low signal-to-noise ratios. On the other hand, overly dense packing of oligonucleotides onto a solid surface causes spatial hindrance. This problem may be even more formidable with longer biomolecules such as cDNAs. Hybridization yields can be increased by up to two orders of magnitude by introducing spacers between the surface and the oligonucleotides ( ). The longer the spacer, the better the hybridization, but, interestingly, there is an optimal spacer length beyond which hybridization yield declines ( ; ). For example, a 40-carbon atom spacer provides a reported 150-fold hybridization enhancement ( ). The density of oligonucleotides is approximately 0.1 pmol/mm ² on a glass surface, two orders of magnitude less than on aminated polypropylene. Thus, a potential advantage of glass over plastic matrices at present is an optimal oligonucleotide density associated with lower spatial hindrance ( ). Improved surface chemistries may one day allow production of arrays on plastic sheets or films, which may dramatically reduce production costs. To this end, film-based chip arrays have recently been described ( ).

The terminal composition (5′ end) of the oligonucleotides influences their duplex yield as measured by relative intensity. As expected, G:C-rich 5′ extremities lead to a better yield than their homologues (same composition but different sequences) ( ). Approaches to modify the 5′ end of oligonucleotides (e.g., covalent addition of a degenerated linker) can therefore be considered to minimize this contribution. Alternatively, knowledge that mismatches at the ends are less destabilizing and therefore more difficult to discriminate by hybridization ( ) has led to clever improvements for detection of hybridization events. Because polymerases and ligases are most sensitive to terminal (as opposed to internal) mismatches, enzymatic methods have been developed with improved detection stringency (solid-phase minisequencing [ ; ; ], genetic-bit analysis [ ], or ligation-assays [ ; ]).

Commercially available arrays have been previously reviewed by . Fabrication of microarrays falls into two broad categories: those made by direct probe delivery onto the solid surface and those made by in situ synthesis. A third method, which was developed by Nanogen (San Diego), consists of prefabricated oligonucleotides captured onto electroactive spots on silicon wafers ( ; ; ; ; ). Modification of the electric field through independent, spatially addressable electrodes can speed up hybridization and, when the polarity is reversed, provide stringent washing ( ). Recently, large single-stranded, circular-sense molecules were used as probes for DNA microarrays and showed stronger binding signals than those of PCR-amplified cDNA probes ( ).