Specificity of DNA microarray hybridization: characterization, effectors and approaches for data correction

Abstract

Microarray-hybridization specificity is one of the main effectors of microarray result quality. In the present review, we suggest a definition for specificity that spans four hybridization levels, from the single probe to the microarray platform. For increased hybridization specificity, it is important to quantify the extent of the specificity at each of these levels, and correct the data accordingly. We outline possible effects of low hybridization specificity on the obtained results and list possible effectors of hybridization specificity. In addition, we discuss several studies in which theoretical approaches, empirical means or data filtration were used to identify specificity effectors, and increase the specificity of the hybridization results. However, these various approaches may not yet provide an ultimate solution; rather, further tool development is needed to enhance microarray-hybridization specificity.

Figure 1.

Illustration of the different levels of specificity of microarray hybridization. (A) Specificity at the probe level; matching between a single probe molecule and a single target molecule: perfect match (i), low match (ii), no match (iii). (B) Specificity at the spot level; matching between multiple probe molecules that make up one spot and multiple different (i.e. derived from more than one reference sequence) target molecules: perfect match (i, red spot), low match (ii, pink spot), no match (iii, gray spot) or cross-hybridization (iv, yellow spot). (C) Specificity at the spot-set level; matching between multiple spots that represent the same reference sequence, but through its different segments, and multiple different target molecules: perfect match (i), low match (ii), no match (iii) or cross-hybridization (iv). Black lines denote probe molecules, red lines denote target molecules of a single reference sequence that perfectly match the spot probes, pink lines denote target molecules of a single reference sequence with lower sequence similarity to the spot probes, green and blue lines denote target molecules of other reference sequences. (D) Specificity at the microarray level; matching between multiple spot-sets and multiple different target molecules: perfect match (red spot), low match (pink spot), no match (gray spot) or cross-hybridization (yellow spot).

Figure 2.

A schematic representation of various effectors of microarray-hybridization specificity, divided into categories, and their interconnections. Numbers is brackets refer to reference numbers. Associations between categories are provided in the text. Several examples for within-category interconnections (designated by arrows): within the category of ‘kinetics of hybridization’: temperature of hybridization or posthybridization affects the kinetics of complex generation, and the duration of hybridization or posthybridization needed for increased specificity. Within the category of ‘sequence of probe and target molecule’: the longer the probe, the more probe-target molecule pairing is expected. As a result, more matching in various positions is expected (13).