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Scientists Can Study an Organism's Entire Genome with Microarray Analysis

A microarray looks like a square grid of spots; each spot represents a different DNA probe sequence. This grid is 20 spots by 20 spots. Spots are colored red, green, yellow, or black, depending on which samples bound to each probe. Red or green spots indicate binding of one sample. Yellow spots indicate binding of both samples. Black spots indicate binding of neither sample. The brightness of the color indicates expression level. In this microarray, there are very few bright red and green spots. Most of the spots are from light yellow to bright yellow. There are also very few black spots.

To compare all the genes of one organism to those of another organism, we must first know how to define the entire gene sequence of each organism. However, looking at all of an organism's genes can be quite daunting. Sometimes, a better option is to consider only those genes expressed by an organism, because these genes may represent just a portion of all the genetic material that the organism contains. That is, an organism may only use a small fraction of its entire genetic sequence, otherwise known as its genome.

What is the genome? What is genomics?

The genome is the set of all genes, regulatory sequences, and noncoding information within an organism's DNA. Thus, the study of genomics considers all of the genetic material contained within and shared between organisms. As genomic techniques advance, researchers continue to accumulate vast amounts of information about DNA sequences from scores of organisms. Indeed, genomic analysis methods enable researchers to analyze genetic data on a scale never before seen in the biological sciences. One genomic technique that has been widely used for large-scale genome comparisons is the DNA microarray.

What is a DNA microarray?

Whereas Northern blots allow scientists to measure the expression of one or several genes at a time, DNA microarrays permit gene expression analysis on a massive scale. In fact, microarray analysis enables researchers to look at expression patterns across all of the genes in an entire genome — and to do so in a single procedure. As a result, it is now possible to monitor the activity of tens of thousands of genes simultaneously.

Microarrays are particularly useful when researchers know that certain genes are being transcribed into mRNA, but they aren't sure exactly what those genes are. For example, scientists know that the expression of certain genes differs depending on environmental conditions, but how can they directly observe which genes vary under which conditions? In short, they can use microarray analysis.

How does microarray analysis work?

In order to conduct microarray analysis (and therefore determine which genes in a sample are active), researchers must first isolate mRNA from a target sample, convert it into complementary DNA (cDNA), and label the cDNA with a fluorescent dye. The fluorescently labeled cDNA is then added to a glass slide or silicon chip upon which thousands of tiny dots of single-stranded DNA have been arranged in a grid pattern. No bigger than the period at the end of this sentence, each dot of DNA in the grid corresponds to a different gene. If any fluorescent cDNA binds to any one of these dots, researchers know that the corresponding genes are active in the sample. But how, exactly, does this process work? To better understand how microarray analysis is carried out, consider the example experiment described in the following sections.

Growing bacteria under two different conditions

An illustration shows two petri dishes lying side-by-side on a flat surface. The petri dish at left contains a purple-colored medium, with streaks of bacteria on top of the medium. The bacteria in this dish were grown at normal temperature. The petri dish at right contains a pink-colored medium, with streaks of bacteria on top of the medium. The bacteria in this dish were grown at a high temperature.

Figure 1: The two temperature conditions for E. coli: normal (left) and heat-exposed (right).

Gene expression in colonies of E. coli bacteria can change when these colonies are exposed to short periods of intense heat. But exactly how do the patterns of gene expression differ? What genes are expressed under one condition, and not the other? The best way to go about answering this question is to do a microarray analysis of the genomes in each experimental condition: normal temperature and heat-exposed. The first step is to create the two conditions by exposing one colony of E. coli to normal temperatures and the other colony to a short burst of high temperature (Figure 1).

Converting RNA

A schematic illustration shows the synthesis of six CDNA strands from six MRNA template strands. The three strands on the left represent unheated samples, and the three strands on the right represent heated samples. For both heated and unheated samples, the top CDNA strand is still undergoing synthesis. The rest of the strands are complete. For both the MRNA and CDNA strands, gray horizontal cylinders represent the phosphate-sugar molecules, and vertical rectangles represent the nitrogenous bases. The nitrogenous bases on the MRNA strand are green, blue, orange, or yellow, representing different chemical identities of the bases. In the CDNA strand, the yellow bases have been replaced by red bases, indicating the switch from uracil in MRNA to thymidine in CDNA. The CDNA strands have been fluorescently labeled; the unheated samples have a green fluorescent label, and the heated samples have a red fluorescent label.

Figure 2: mRNA is converted to fluorescently-labeled cDNA.

After this treatment is complete, the bacterial cells are removed from both culture plates and mRNA is extracted from them. Reverse transcriptase and fluorescently-labeled nucleotides are then added to the two test tubes containing the extracted mRNA. Specifically, each tube receives nucleotides marked with a particular fluorescent color: red or green. By using one fluorescent color for the tube of normal RNA and another color for the heat-exposed RNA, researchers can follow the genetic material from each colony during later stages of analysis.

Within each test tube, the newly synthesized, fluorescently-labeled cDNA strands form complementary DNA strands with the original mRNA strands (Figure 2). Next, the mRNA is specifically degraded so that only the cDNA copy of the mRNA message is left behind. At this point, the cDNA that was synthesized in each tube is associated with its corresponding color (red or green). Remember, this sample-specific labeling means that the scientists will easily be able to track which cDNA came from normal cells and which cDNA came from heat-exposed cells during later phases of the microarray process.

Applying solution to the DNA chip

A schematic shows four free-floating, single-stranded CDNA molecules and two CDNA molecules that have bound to complementary DNA probes on a microarray. Gray, horizontal cylinders represent the deoxyribose sugar molecule, and vertical rectangles represent the nitrogenous bases. The nitrogenous bases are blue, red, green, and orange, representing different chemical identities of the bases. In two of the single-stranded molecules, green circles are bound to each deoxyribose sugar molecule, representing a green fluorescent label. In the other two single-stranded molecules, red circles are bound to each deoxyribose sugar molecule, representing a red fluorescent label. The two bound CDNA strands are also fluorescently labeled; one strand is labeled green, and the other strand is labeled red.

Figure 3: Fluorescently-labeled cDNAs from the normal and high temperature samples bind to complementary DNA strands on the DNA chip.

Next, the fluorescently-labeled cDNA samples from normal and heat-exposed cells are combined into a single solution and applied to a microarray chip, also known as a DNA chip. The DNA chip is covered with a grid of small dots, each with multiple single-stranded pieces of DNA attached to it. Each single strand represents a specific gene sequence.

After the cDNA is applied to the microarray chip, the cDNA molecules will bind to any complementary strands that exist on the chip (Figure 3). As different genes are on different dots, some of the cDNA in the sample binds to certain dots, some binds to other dots, and some does not bind to any dots whatsoever. This binding identifies which genes were expressed in the original bacterial colonies, because the bound cDNA is joining with partner strands that are already preprogrammed onto the microarray chip. Then, any unbound cDNA is washed away from the chip with a careful rinse, so the only cDNA molecules left on the chip are those that found complementary partners on the chip and bound with them.

Imaging the chip

A microarray chip looks like a 14 by 14 grey grid of spots. Most of the spots are white, but some of the spots are red. A red gradient overlaying the microarray chip, which is clear on the left side and progressively darker red as the figure progresses to the right, indicates that the microarray is being scanned with a laser that detects red fluorescence.

Figure 4: Scanning the microarray chip.

The chip is then scanned with a special laser that detects the fluorescent molecules attached to each cDNA strand. A single dot will "light up" if cDNA is attached to a complementary sequence on that dot (Figure 4). Here, because of the sample-specific fluorescent labeling, green dots reflect genes that are highly expressed in the normal temperature sample, and red dots reflect genes that are highly expressed in the heat-exposed sample. When red and green fluorescent molecules exist in equal amounts on the same dot, the dot will appear yellow, so yellow dots reflect genes that are expressed at equal levels in normal and heat-exposed samples.

Analyzing the imaged data

Sixteen microarray chips are arranged in a four by four square diagram. Each chip looks like a 14 by 14 grid of spots. Spots are colored either red, green, yellow, white, or gray.

Figure 5: Multiple gene chips are needed to scan the entire genome for expression patterns.

Because each gene chip comes with a map of the genes represented by each dot, the pattern of green, red, and yellow dots can be easily translated into gene names. In addition, a computer connected to the scanning light can detect and measure the intensity of the color at each dot. By comparing the intensity of the fluorescent signals, researchers can estimate the relative abundance of each mRNA transcript. Moreover, because each chip used in this experiment surveys 6,000 different genes, the experiment can be repeated using different gene chips until every gene in the bacterium has been surveyed (Figure 5).

Consequently, the raw data generated from multiple microarray chips look like sparkling patterns of red, green, and yellow dots. For the above experiment with bacterial colonies, these data tell a story of gene expression across the entire E. coli genome under two different environmental temperatures (Figure 6).

Forty-eight microarray chips are arranged in a four by twelve rectangle. Each chip is a square grid of spots. The individual spots are too small to detect, but the overall color of the chips is varying shades of mustard yellow.

Figure 6: A photograph of real microarray chip data, arranged in a grid. Multiple chips, such as the ones shown here, reveal expression data for an entire genome.