An oligonucleotide microarray is a sophisticated molecular tool that allows for the simultaneous measurement of the activity levels of thousands of genes. This technology provides a powerful snapshot of the genetic state of a biological sample, revealing which genes are switched on or off under specific conditions. By miniaturizing and parallelizing genetic analysis, microarrays have become a foundational technique for understanding complex biological processes, from disease progression to cellular responses.
Defining Oligonucleotide Microarrays
The technology is built upon a solid, non-reactive surface, typically a glass slide or a silicon chip, which acts as the physical platform for the experiment. This surface is engineered to hold a vast number of microscopic spots, each representing a specific chemical reaction site. The molecules fixed to these spots are known as probes.
These probes are synthetic, single-stranded DNA molecules called oligonucleotides, which are short sequences. Each probe is designed to be a perfect match for a specific, known genetic sequence. Thousands of these sequences are either spotted onto the slide using robotic machinery or chemically synthesized directly onto the surface.
The arrangement of these probes forms a highly organized grid where the location of every spot corresponds to a known gene. These fixed oligonucleotide probes act as molecular traps, ready to capture and bind to any complementary genetic material present in a biological sample.
The Operational Process
The process begins with preparing the biological sample, often involving the extraction of messenger RNA (mRNA) from the cells being studied. Because mRNA is unstable and cannot be directly measured by the array, it must first be converted into a more stable form called complementary DNA (cDNA) using an enzyme called reverse transcriptase. This cDNA is the target molecule that will be tested against the array.
A distinctive step in sample preparation is the labeling of the target cDNA molecules with fluorescent dyes, which serve as molecular beacons. In a common experimental design, two different samples—such as a healthy tissue sample and a diseased tissue sample—are compared simultaneously. Each sample is labeled with a different colored fluorescent dye, such as a red dye for one sample and a green dye for the other.
The labeled target molecules are then applied to the surface of the microarray chip, a stage known as hybridization. During this step, the single-stranded target molecules will naturally seek out and bind to their complementary oligonucleotide probes fixed on the array, following the fundamental rule of nucleic acid base pairing. The stringency of the conditions is carefully controlled to ensure only highly specific binding occurs.
Following hybridization, the slide is thoroughly washed to remove any target molecules that did not bind or that bound non-specifically to the probes. This washing step is important for minimizing background noise. The final step involves placing the processed array into a specialized laser scanner.
Data Acquisition and Analysis
The scanner systematically illuminates each spot on the array with a laser, which excites the fluorescent dyes on the bound cDNA molecules. A detector measures the resulting light emission at each spot, quantifying the intensity of the fluorescence.
A spot showing only red fluorescence indicates that the gene represented by that probe was highly active only in the red-labeled sample. Conversely, a spot that appears yellow (a combination of red and green light) means the gene was active in both samples at similar levels. The intensity data is then quantified by software, providing a numerical measure of gene activity for every gene on the chip.
Key Scientific Uses
The data generated from a microarray experiment is primarily used for gene expression profiling. This application involves comparing the activity of thousands of genes between two or more different conditions, such as cells treated with a drug versus untreated cells, or cancerous versus normal cells. By identifying genes that are significantly turned up or down, researchers gain insights into the underlying molecular mechanisms of biological phenomena.
Microarrays are also a powerful tool for genotyping, which is the process of determining the genetic makeup of an individual organism. They can detect single-nucleotide polymorphisms (SNPs), which are tiny single-base variations in the DNA sequence that contribute to genetic diversity. By using probes that match different versions of an SNP, the array can quickly determine an individual’s genotype at thousands of sites across the genome.
In drug discovery and toxicology, microarrays are employed to understand how cells respond to new pharmaceutical compounds. Measuring the change in gene expression patterns after drug exposure can help predict the drug’s effectiveness and identify potential side effects. This high-throughput screening capability accelerates the early stages of drug development.