Purpose / What It Accomplishes #
DNA microarrays, often referred to as gene chips, are high-throughput molecular biology tools designed for the simultaneous measurement of expression levels for thousands of genes, or for detecting specific DNA sequences, in a single experiment. They enable researchers to perform comparative analysis of gene expression profiles under different biological conditions, such as healthy versus diseased states.58
Principle / Theoretical Basis #
The underlying principle of DNA microarrays is nucleic acid hybridization. Thousands of microscopic spots, each containing a unique, known single-stranded DNA probe (which can be either a synthetic oligonucleotide or a complementary DNA (cDNA) fragment), are precisely immobilized in an ordered array on a solid surface, typically a glass slide.58 Fluorescently labeled target DNA or RNA (specifically, cDNA synthesized from messenger RNA (mRNA) of a sample) is then introduced to the array. Due to the principle of complementary base pairing, the labeled target sequences will hybridize (bind) only to their complementary probes on the chip. After stringent washing to remove unbound molecules, the intensity of the fluorescent signal emitted from each spot is directly proportional to the amount of target sequence present in the original sample, allowing for relative quantification of gene expression.58
Step-by-Step Explanation #
- Equipment and Reagents Required: A DNA chip (microarray slide); a microarray scanner (comprising a laser, a camera, and a computer for data acquisition); biological samples (e.g., cells or tissues from a control group and an experimental group); reagents for RNA extraction; reverse transcriptase enzyme; fluorescent dyes (e.g., Cy3 and Cy5 for two-color arrays); hybridization buffer; and various wash buffers.58
- Workflow from Start to Finish:
- Sample Collection: Biological samples are collected from different conditions (e.g., healthy cells/tissues versus diseased or treated cells/tissues) to allow for comparative analysis.58
- mRNA Isolation: Total RNA is extracted from the collected samples. From this total RNA, messenger RNA (mRNA) is specifically isolated, often using oligo-dT column beads that selectively bind to the poly-A tail characteristic of eukaryotic mRNA. During this process, it is crucial to inactivate RNases to prevent RNA degradation.58
- cDNA Synthesis and Labeling: The isolated mRNA from each sample is reverse transcribed into complementary DNA (cDNA) using reverse transcriptase. During this synthesis, fluorescent dyes are incorporated into the cDNA. Typically, two different fluorescent dyes (e.g., green-fluorescent Cy3 for control samples and red-fluorescent Cy5 for experimental samples) are used to distinguish between the samples.58
- Hybridization: The fluorescently labeled cDNA samples (e.g., mixed Cy3- and Cy5-labeled cDNA) are combined and applied to the microarray chip. The chip is then incubated under specific hybridization conditions (temperature, time, buffer) to allow the labeled cDNA molecules to bind to their complementary probes immobilized on the array.58
- Washing: After hybridization, the chip undergoes a series of stringent washes to remove any non-specifically bound or unhybridized labeled cDNA. This step is critical for reducing background noise and ensuring the specificity of the signal.58
- Scanning: The hybridized and washed microarray chip is placed into a microarray scanner. The scanner uses a laser to excite the fluorescent dyes on the hybridized spots, and a high-resolution camera captures the emitted fluorescence from each spot. The computer records the signal intensities for each dye at each spot.58
- Data Analysis: The scanned images are analyzed using specialized bioinformatics software. The intensity of fluorescence at each spot (and the ratio of intensities for two-color arrays) indicates the relative expression level of that gene. The software identifies differentially expressed genes, and various quality control checks are performed to ensure data reliability.58
Variations / Modifications #
Microarray technology has evolved into various formats for different applications:
- cDNA Microarrays: Utilize longer complementary DNA strands (cDNA fragments) as probes, typically generated by PCR and spotted onto the slide.58
- Oligo DNA Microarrays: Employ shorter, chemically synthesized oligonucleotide probes, often synthesized directly onto the chip surface.58
- BAC Microarrays: Use bacterial artificial chromosome (BAC) clones as probes, often for comparative genomic hybridization (CGH) to detect large-scale DNA copy number variations.58
- SNP Microarrays: Designed to detect single nucleotide polymorphisms (SNPs) across a genome for genotyping applications.58
- Beyond DNA, the microarray concept has been extended to other biomolecules, leading to protein microarrays, peptide microarrays, tissue microarrays, cellular microarrays, chemical compound microarrays, and antibody microarrays, each adapted for specific analytical purposes.59
Applications #
DNA microarrays have broad applications in genomics research and diagnostics. They are widely used for gene expression profiling, enabling researchers to understand how gene activity changes in response to various stimuli, diseases, or developmental stages. They are applied in the diagnosis of pathogenic and genetic diseases, identifying specific microbes in environmental samples, and genotyping genomes through SNP analysis.58 Furthermore, microarrays are used to detect DNA mutations, study genomic gains and losses, aid in drug discovery by identifying drug targets, and contribute to toxicological research by assessing gene expression changes in response to toxins.
Strengths and Limitations #
- Strengths: Microarrays offer high-throughput analysis, allowing the simultaneous examination of thousands of genes or sequences in a single experiment, providing a broad overview of gene expression. They are capable of generating data for many genes in a relatively short time, offering insights into dynamic biological processes. The technology is well-established and versatile for various applications.59
- Limitations: A significant limitation is that microarrays can only detect sequences for which probes were specifically designed, meaning they require a priori knowledge of the genes of interest and may miss novel transcripts or unexpected variations.1 They can suffer from problematic cross-hybridization artifacts, where highly similar sequences bind to the same probe, leading to reduced specificity. Their ability to accurately quantify very low or very highly expressed genes can be limited. The technology can be expensive, especially for custom or commercial arrays, and the large volume of data generated requires substantial processing time and complex interpretation.58 DNA chips also have a limited shelf life due to probe degradation.59
Why It Should Be Learned #
Microarrays were a pioneering high-throughput technology that fundamentally transformed gene expression analysis and large-scale genomic studies. While newer sequencing technologies like RNA-seq have emerged, microarrays remain relevant for specific applications and provide a crucial historical context in the evolution of genomics. The transition from microarrays to next-generation sequencing highlights a significant shift in genomics from a hypothesis-driven approach to a more discovery-driven paradigm. Microarrays, by design, are tailored to test specific hypotheses about known genes. In contrast, RNA-seq offers the capability to discover novel transcripts and provide a more comprehensive view of the transcriptome without predefined biases, leading to a more complete understanding of biological systems. Understanding this evolution is key to appreciating the advancements in genomic research.