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Applications of DNA microarrays in chemical biology
Planar surfaces functionalized with single- or double-stranded nucleic acids have enabled researchers to address a variety of salient biological and biochemical questions in recent years. The general architecture of modern DNA microarrays reflects the historical progression from the sequence-specific probing of whole chromosomes immobilized on glass slides (as early as 1961 with fluorescent in situ hybridization) and the low-density porous membrane arrays available since the early 1990s, to the high-density (102-104 features/mm2) solid support platforms that exist today. The massively parallel processing capabilities of these picomolar-range contemporary arrays provide for the generation of large data sets and multiplexed analysis. Furthermore, several top-down and bottom-up assembly methodologies provide researchers with the option for “in-house” production of arrays from custom oligonucleotide libraries or the use of commercial genome chips, notably those developed by Affymetrix and Agilent Technologies. DNA microarrays can be used to conduct several general types of experiments, most of which rely on the hybridization of fluorescently-labeled single-stranded DNA molecules isolated from a biological sample to their single-stranded complement probes presented on an array. One of the earliest conceived applications for DNA microarrays was for single-nucleotide polymorphism (SNP) genotyping. Since SNPs are a “quick and dirty” approach to detect genetic indicators of pathologies and lineages, arrays theoretically provide a facile method for diagnosis; this was confirmed experimentally in the late 1990s in the successful SNP analysis of human tumors. Although there are currently commercially available arrays (e.g. |Affymetrix Bovine Mapping chips) to characterize SNPs, it seems likely that the nascent availability of high-throughput and low-cost pyrosequencing will become the preferred method of recognition, or replace the need for SNP detection altogether with rapid whole-genome sequencing.

A different application of microarray technology that has become the gold standard for RNA analysis in recent years is the widespread utilization of expression microarrays, or “gene chips”. Gene chip preparation calls for the quantitative reverse transcription of the total cellular RNA pool into labeled and fragmented single-stranded DNA prior to hybridization-based capture. Up- and down-regulation of genes in response to stressors or disease states are quantitatively compared in cell lines and organisms. Coupled expression microarray and quantitative proteomics experiments have allowed for the in-depth exploration of the oftentimes non-linear relationship between the abundance of a particular transcribed message and that of its corresponding translated protein. These integrative studies, partially enabled by quantitative DNA microarray technology, have been successfully applied to a variety of biological systems, including yeast, bovine, mouse, bacterial, and human. The expression analysis community has amassed such a significant amount of expression microarray data that they are freely available in public databases.

These types of surfaces can also be used to analyze DNA-protein interactions on a genome-wide scale via chromatin immunoprecipitation, followed by an array-based analysis of the DNA (ChIP-chip). ChIP-chip experiments are enabled by the co-purification of a DNA-binding protein of interest with its corresponding genomic loci when a cross-linked chromatin extract is probed with an antibody to said protein. After purification, amplification and labeling, the DNA is applied to a microarray representing the entire genome; the data are plotted as a histogram that resolves the specific genomic regions associated with that protein. ChIP-chip experiments have provided the scientific community with a wealth of information about the steady-state genomic locations of DNA-binding proteins, such as histones, transcription factors, and polymerase machinery, and have also been successfully applied to studies on the dynamics of transcription factor binding. The data from these experiments may be further manipulated to computationally derive consensus binding sequences for some transcription factors, giving the opportunity for insight into the in vivo behavior of the factor, deeper than simple information about localization.

DNA microarrays are also amenable to the direct analysis of protein-DNA interactions in kinetic binding assays as analyzed by surface plasmon resonance (SPR). This experimental approach also relies on single-stranded DNA immobilized on a high-density array; however, the quantitative readout is based on a change in the optical properties of the DNA-functionalized surface when a protein flowed over the surface binds to the sequence in a particular surface feature. DNA-functionalized arrays analyzed with SPR in this way have yielded kinetic data regarding fundamental molecular biological processes. Recently, SPR analysis of a DNA microarray and components of the DNA replication machinery helped to elucidate the biochemical nuances of the replication fork.

High-density DNA microarrays have emerged as an important component of the chemical biology toolkit. The existing technology allows for the construction of customizable, as well as general, arrays and provides researchers with the opportunity to generate robust data from many different types of biological inputs. Considering the relatively recent shift in the scientific community away from binary perturbation/readout studies and toward “big science” and large data sets, it seems likely that DNA microarrays will continue to enable pertinent biological research for many years to come.

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