DNA Microarrays

A DNA microarray consists of thousands of single-stranded DNA probes arranged in a grid on a glass or silicon chip. Each probe is covalently attached to the chip surface, forming an array of spots where each site represents a single target gene. This arrangement allows one microchip to analyze tens of thousands of genes simultaneously, making it a powerful high-throughput tool for genomic analysis.

Total mRNA from cells is isolated and converted to fluorescently labeled cDNAs. When exposed to the microarray, these cDNAs hybridize with complementary probes. The fluorescent signal intensity at each spot is directly proportional to the amount of bound cDNA, allowing software to quantify gene expression levels by comparing relative concentrations between samples.

In dual-color analysis, a reference sample is labeled with a green fluorophore while a test sample is labeled red. If test expression is lower than reference, the spot appears green; higher expression appears red. Yellow indicates equal expression levels in both samples, enabling direct visual comparison of gene expression differences between two conditions.

Microarray probes are single-stranded DNA segments corresponding to each gene's coding region. Probe nucleotides can be copied from genomic DNA using PCR or synthesized directly onto the chip's surface. These probes are then covalently attached to the chip through surface functionalization, typically via chemisorption of silanes to silica surfaces, creating stable, chemically resistant probe arrays.

DNA microarrays detect transcription factor binding on chromatin and identify single-nucleotide variations in genomic DNA. They also monitor microRNA expression, measure DNA copy numbers, and detect single-nucleotide polymorphisms. Commercially, microarrays are used in biotechnology, forensics, toxicology, drug discovery, and targeted therapy for infectious and genetic diseases.

DNA microarray probes are designed to complement specific target cDNA sequences, so the mRNA sequence must be known beforehand. This prerequisite makes microarrays unsuitable for discovering novel mRNA sequences. For such applications, researchers use alternative methods like high throughput sequencing for transcriptome studies, which can identify unknown sequences without prior sequence knowledge.

DNA microarrays are the most popular microarray technology because DNA probes are chemically stable and retain their native interactions despite harsh chemicals and high temperatures used during probe loading and chip preparation. This stability, combined with their ability to analyze thousands of genes in parallel and their relatively low cost, makes DNA microarrays ideal for genome-wide expression studies.