How do DNA microarrays work?

DNA microarrays work on the principal of base-pairing . Base-pairing allows probes to hybridize to targets on the microarray.

At a basic level microarrays are implemented as follows: a cell's RNA is extracted. This RNA (targets) is then multiplied, labeled with fluorescence and hybridized to existing DNA (probes) on the microarray. After hybridization, the probes that were hybridized with targets are fluorescent and a computer scanner is able to detect this fluorescence. Those probes that are fluorescent correspond to the genes that were expressed in the cell.

The microarray is composed of millions of spots (sometimes referred to as cells), each with thousands of probes. Each 20 micrometer cell can contain up to 10^7 probes. The enormous number of probes is to increase hybridization probability and possibilities.

Figure 1 is an illustration of the principal of hybridization. A cell (G) is laced with probes of DNA (either oligonucleotide sequences, or cDNA. See below for more information). The fluorescent targets (green circles) are then exposed to the microarray and allowed to hybridize.



Detailed cDNA Microarray Technology Scheme

Experiments using cDNA microarrays typically involve two cells: a control cell and an experimental cell. First, using robotics, the microarray is laced with DNA probes corresponding to the genes of interest (or the entire organism's genome).

Second, mRNA from a control cell and an experimental cell is isolated. This mRNA corresponds to the genes in the cell that are being expressed (See basic biology, Why study genes). Using reverse transcriptase , the mRNA is then converted to cDNA . The cDNA from both cells are labeled, using fluorescence, different colors. This fluorescent dye can be identified by a computer scanner. The labeled cDNA is considered the target, which hybridizes via base-pair interactions with the probe.

Once the targets are exposed to the microarray for a sufficient amount of time to allow for hybridization (typically 12-16 hours), the array is washed. Certain probes on the array will now be fluorescent, because the fluorescent targets have hybridized to them.

Only the probes containing genes that have been transcribed in the cell will be fluorescent. Those genes that are being expressed in the control cell will fluoresce one color (green), while those expressed in the experimental cell will fluoresce another color (red). Those genes expressed in both cells will have a mixed color (yellow). The amount each gene is being expressed can also be measured by how intense the fluorescence is.

A computer is used to measure the intensity and color of each and every spot on the microarray. Software can then be used to produce data on exactly which genes are being expressed in the cells, and how much each of those genes is being expressed.

Oligonucleotide Arrays

Oligonucleotide arrays are commonly used in biology laboratories and in clinical research projects. The Affymetrix GeneChip is the most widely used oligonucleotide array. Whereas the aforementioned cDNA microarrays use long strands of DNA as fixed probes, the GeneChip uses oligonucleotide sequences as its probe. The whole genome of an organism can be placed on a single microarray as oligonucleotide probes. These oligonucleotide sequences are usually around 25 base pairs in length . Below is a explanation of how Affymetrix's technology works.

                    Detailed Affymetrix GeneChip Scheme

The basic procedure for using an Affymetrix array is very similar to a cDNA array. Initially, Affymetrix GeneChips are laced with oligonucleotides using a combination of photolithography and combinatorial chemistry. The GeneChip is then sold to researchers, who perform their experiments.

In order to use the array, first mRNA is extracted from a cell and reverse transcriptase is applied to obtain cDNA. In oligonucleotide arrays, in vitro transcription (for more information on in vitro transcription, occurs to obtain biotin labeled cRNA (See Basic Biology: Base Pairing with RNA). These cRNA molecules are then exposed to the microarray. Overnight, the cRNA molecules (the targets) hybridize to the oligonucleotide probes. After hybridization, the chip is stained with a fluorescent molecule (streptavidin-phycoerythrin) that binds to biotin. The staining protocol includes a signal amplification step that employs anti-Streptavidin antibody and biotinylated goat IgG antibody (The series of washes and stains with aforementioned reagents binds the biotin and provides an amplified flour that emits light when the chip is then scanned with a confocal laser and the distribution pattern of signal in the array is recorded) A scanner analyzes the GeneChip for signals. Advanced algorithms are then used to give data on the expression levels of the genes of interest.