An oligonucleotide microarray is a miniature biological laboratory on a solid surface, often the size of a microscope slide. This “gene chip” functions as a tool for measuring the activity of thousands of genes at once, containing a collection of short, synthetic DNA sequences. This allows scientists to get a snapshot of which genes are turned on or off within a cell at a specific moment. By seeing the complete pattern of gene activity, researchers can identify networks of genes working together and better understand complex biological systems, such as the changes that occur when a cell becomes diseased.
Building the Microarray Chip
The foundation of a microarray is the precise placement of thousands of distinct oligonucleotide probes onto a solid substrate. An oligonucleotide is a short, single-stranded molecule of synthetic DNA with a sequence designed to match a specific gene. The surface, usually a glass slide or silicon chip, is chemically treated to allow these probes to be permanently attached in an orderly and mapped grid, where each spot represents a different gene.
The creation of these chips can be accomplished through methods like photolithography, which uses light to direct the synthesis of oligonucleotides on the chip’s surface. This technique allows for extremely dense arrays. Another method involves using a robotic device to “spot” pre-synthesized oligonucleotides onto the glass slide. The final product is a chip with a uniform concentration of probes at each spot, which helps ensure reliable results.
The Experimental Workflow
The process begins with the collection of biological samples, for instance, cells from a cancerous tumor and from healthy tissue of the same patient. From these samples, scientists extract messenger RNA (mRNA), the molecule that carries genetic instructions from DNA to the cell’s protein-making machinery. The amount of a specific mRNA molecule is a direct indicator of its corresponding gene’s activity level.
Because mRNA is unstable, it is converted into a more durable molecule called complementary DNA (cDNA) through reverse transcription. During this step, the cDNA is tagged with fluorescent molecules. For example, the cDNA from cancerous cells might be labeled with a red fluorescent dye, while cDNA from healthy cells is labeled with a green one, allowing for a direct comparison.
The labeled cDNA solutions are then combined and washed over the microarray chip. This step is known as hybridization, where the single-stranded cDNA molecules find and bind to their complementary oligonucleotide probes on the chip. A cDNA molecule will only stick to the spot whose probe has a matching genetic sequence, based on the rules of DNA base-pairing.
After allowing time for hybridization, the chip is washed to remove any cDNA that has not bound to a probe. This ensures that the only fluorescent signals detected are from cDNA molecules that have successfully paired with their counterparts on the array. The chip is now ready for scanning.
Reading and Analyzing the Data
The microarray chip is placed into a specialized scanner that uses lasers to illuminate the fluorescent tags attached to the cDNA. The scanner excites the dyes at each spot, and detectors measure the intensity and color of the light emitted from every probe location. This process generates a high-resolution image of the chip, revealing a pattern of colored dots.
The color and brightness of each spot provide specific information about gene activity. A bright red spot indicates the gene was highly active in the cancerous cells, while a bright green spot signifies high activity in the healthy cells. A yellow spot means the gene was active in both samples, and a dark spot indicates little to no activity in either.
To visualize these results, the data is often presented as a heat map, which is a graphical representation where colors denote the level of gene expression. For example, red might represent high expression and green might represent low expression. This allows researchers to quickly identify patterns and see which groups of genes are behaving differently between the two conditions.
Key Uses in Research and Diagnostics
Oligonucleotide microarrays have several applications in research and diagnostics:
- Gene expression profiling: Researchers can compare diseased tissue with healthy tissue to identify which genes are “turned on” or “turned off” by a disease. This provides insights into the molecular mechanisms of a condition and can reveal how cells respond to drug treatment.
- Disease diagnosis and classification: Different types of cancer, which may appear identical under a microscope, can have distinct genetic signatures. Microarray analysis reveals these underlying differences, helping to classify tumors into more specific subtypes for more effective, targeted treatments.
- Drug discovery and development: By observing which genes are affected by a disease, scientists can pinpoint proteins that may be suitable for drug intervention. The technology can also test the effects of candidate drugs on a genomic scale to understand their mechanisms and potential side effects.
- Detection of genetic variations: Microarrays can detect single nucleotide polymorphisms (SNPs), which are tiny variations in the genetic code between individuals. Identifying these SNPs can help predict disease risk and personalize medicine by tailoring treatments to an individual’s genetic makeup.