DNA MICROARRAY ANALYSIS

Introduction

DNA microarrays, also sometimes referred to as 'gene chips,' are among the most powerful new tools in the field of genomics-based biotechnology. DNA microarray analysis overcomes many of the problems of traditional molecular techniques that look at genes and their products one at a time. Such methods greatly restrict assay throughput and also typically fail to provide insights into integrated (whole-genome) gene function.

DNA microarray analysis allows researchers to simultaneously monitor the activity of hundreds or even thousands of distinct DNA sequences, to the point where entire genomes might be represented on a single microarray (aka, 'chip'). This approach allows biotech professionals to perform assays that analyze thousands of genes at a time.

As with several other DNA analysis tools, microarray analysis is based on the fundamental principle of DNA base-pair complementarity; specifically, the nucleotide adenine always pairs with thymine, while guanine always pairs with cytosine.

Microarray Basics

A DNA assay array presents a defined set of DNA samples of known sequence, most often a collection of known or suspected gene fragments. It is used to test whether these sequences match those in an unknown DNA sample, based on the degree to which known and unknown DNA strands complement and therefore bind to each other.

If the array consists of sample well spots with a diameter of greater than 300 microns, it is considered a macroarray. The results of macroarray assays can be visualized using standard gel or blot scanners. Microarrays, on the other hand, generally consist of thousands of sample well spots with diameters of less than 200 microns each. Specialized robotics and laser imaging hardware are required to manipulate and visualize microarrays.

Microarrays are fabricated on glass or nylon substrates with the aid of specialized high-speed robotics. To each of the many well spots on the microarray is added a specified DNA probe-an immobilized strand of DNA of known sequence. A sample of target DNA of unknown sequence is added to the array and complementarity (degree of base-pair matching) with each of the probes is measured. Most often the target DNA is cDNA (complementary DNA), which has been laboratory-synthesized via reverse transcriptase enzyme using mRNA (messenger RNA) as a template. Since mRNA is transcribed in vitro by genes and sent to the cell's ribosomes to be translated into proteins, utilizing cDNA synthesized from mRNA ensures that the microarray assay targets represent regions of source genome that are actively coding for gene products.

There are two general types of applications for DNA microarray technology. The most basic of these is gene discovery based on identifying complementary hybridization between target DNA and specific well spots on the array representing documented genes or fragments.

Still more refined, and substantially more versatile, are microarray applications designed to assess the expression level or abundance of genes represented in the cDNA target material. This is usually done through studies of comparative gene expression. Such studies are used in research aimed at disease diagnosis, toxicological research, and in cutting-edge drug discovery programs. Here, DNA microarray analysis is used to compare levels of gene expression in two different populations of cells. Which cell types are compared is up to the researcher, but possibilities include comparing cells from different tissues, healthy cells versus tumor cells, or comparing a control cell population to one that has been subjected to an experimental drug or other environmental stressor.

Natural Products Drug Screening Example

Consider a more detailed example of DNA microarray analysis/comparative gene expression such as might be employed in a marine natural products drug discovery program. In this example, the assay is used to compare cancer cells treated with a drug candidate to untreated cancer cells to pinpoint differences in gene expression between them and to gain a better understanding of drug activity and cell response at the genomic level. This is a strategy marine biomedical researchers are currently using to zero in on the precise mechanism of action of a family of sponge-derived anticancer compounds called the lasonolides.

In the drug-screening scenario, a tumor cell line of interest is selected and a DNA microarray is assembled that includes probes representing gene fragments coding for proteins produced by the tumor cells and putatively involved in disease progression. The tumor cells are split into two populations. One of these acts as a control group while the other is exposed to the natural product being screened as a potential drug therapy against this type of cancer. Then, mRNA from each population is extracted and purified, and this is reverse-transcribed to the more stable cDNA form.

Each of the two resulting cDNA target samples is labeled with a distinct reporter molecule that will later identify (through fluorescence) cDNA presence once bound on the DNA microarray. The labeled cDNA targets are then added to the array. The assay simultaneously tests the ability of target cDNA fractions to hybridize to complimentary sequences on hundreds or thousands of distinct strands of immobilized microarray DNA. Essentially, each spot on the microarray is an assay for the presence of a different cDNA sequence.

After hybridization, unbound cDNA is washed off and the microarray is scanned to see how much cDNA remains bound to the tethered probe DNA at each of the well spots. If target cDNA is present on a well spot it will detectable by its fluorescence when excited by laser light. Emitted fluorescence is produced at distinct wavelengths by the two cDNA samples, and is captured by a detector which records its intensity. The greater the intensity of fluorescence at a well spot, the larger the quantity of target cDNA bound there.

The accompanying image is a scanned microarray in which fluorescent reporter intensity has been false-color converted to show as red and green. The intensity of the color indicates the amount of hybridization (and hence the amount of bound cDNA) at a well spot. Yellow well spots represent probe sites at which bound cDNA samples from both cell populations were present in approximately equal amounts (red + green = yellow). In contrast, well spots tending more decidedly toward either red or green indicate measurably higher levels of cDNA (and thus mRNA) from one or the other cell population.

Through this assay technique, researchers can identify gene sequences in the cancer cell line whose expression is affected by exposure to the drug treatment. Identifying up-regulation or down-regulation of specific genes after drug exposure can provide valuable insights into how the drug in question targets cancer cells. Targeted drug screening such as this holds great potential in the search for next-generation medicines that specifically attack cancer cells but do not exhibit the general cytotoxicity associated with many present chemotherapeutics.

Related Weblinks

DNA Microarray (Genome Chip) - Monitoring the Genome on a Chip (tutorial created by Leming Shi, Ph.D)
http://www.gene-chips.com

Anatomy of a Comparative Gene Expression Study (tutorial site created by Jeremy Buhler)
http://www.geneticengineering.org/jbuhler