15.6 : DNA Microarrays

A DNA microarray contains thousands of single-stranded DNA probes arranged in a grid on a glass or silicon chip. The DNA of interest is fluorescently labeled and can hybridize to its complementary probe on the microarray.

This technique allows the simultaneous measurement of the relative concentrations of specific cDNA sequences.

Commonly, DNA microarrays are used to quantify gene expression by measuring the levels of cDNA corresponding to particular mRNA transcripts.

Other uses include detecting the binding of transcription factors on chromatin or identifying single-nucleotide variations in genomic DNA.

Microarray probes for measuring mRNA are single stranded DNA segments corresponding to each gene’s coding region to be analyzed. The probe nucleotides can either be copied from genomic DNA using PCR,  or synthesized directly onto the chip’s surface.

These probes are covalently attached to a chip forming an array of spots, where each site represents a single target. Therefore, one DNA microchip can analyze tens of thousands of genes.

For a genome-wide analysis, the total mRNA content is isolated from the cells of an organism and converted to fluorescently labeled cDNAs. When this sample is exposed to a DNA microarray chip, the probes pair with their complementary cDNAs.

The fluorescent signal at each spot is directly proportional to the amount of cDNA bound, and can be analyzed by the appropriate software to quantify gene expression.

Although this analysis does not yield the absolute concentrations of the mRNAs, it can be used to compare their relative concentrations— hence gene expression levels, in two samples under different conditions.

A reference sample could be labeled with a green fluorophore, while a test sample would be labeled red.

If the test sample has a lower expression than the reference sample, then the spot appears green, while a higher expression is indicated by a red spot. Yellow indicates equal expression levels in both samples.