DNA Microarray Definition and Principle

Definition: Ordered arrangement of DNA probes on silicon surface is called DNA microarray or gene chip.

A microarray is typically a glass side, on to which DNA molecules are attached at fixed locations as spots.

Principle: Hybridization. It is the property of complementary nucleic acid sequences to specifically pair with each other by forming  hydrogen bonds between complementary nucleotide base pairs.

The cDNA is labelled with reporter molecule (generally florescent molecules) to detect it when bound to microarray.

Under in-vitro conditions RNA is unstable therefore, it is converted into cDNA which is derived from a distinct messenger RNA, each of which represents an expressed gene. The reporter fluorescent molecule emits fluorescence which indicates high expression levels of a particular gene. Probe target hybridization is usually detected and quantified by detection of fluorophore, silver or chemiluminescene labelled targets to determine relative abundance of nucleic acid sequences in the target.

Fluorescently labelled target sequences that bind to a probe sequence generate a signal that depends on the strength of the hybridization determined by the number of paired bases, the hybridization conditions (such as temperature) and washing after hybridization.

Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome.

 

Two colour microarrays or two channel microarrays are typically hybridized with cDNA prepared from two samples to be compared (healthy vs diseased) and that are labelled with two different fluorophores. Florescent dyes commonly used for cDNA labelling Cy3, which has a fluorescence emission wavelength of 570 nm (corresponding to the green part of the light spectrum) and Cy5 with a fluorescence emission wavelength of 670nm (corresponding to the red part of the light spectrum)

The two Cy labelled cDNA samples are mixed and hybridized to a single microarray that is then scanned in a microarray scanner to visualize fluorescence of the two fluorophores after excitation with a laser beam of a defined wavelength. Relatives intensities of each fluorophores may then be used in ratio based analysis to identify up regulated and down regulated genes.

 DNA Microarray Procedure (Steps) and Applications