DNA Microarray Methodology


This animation will demonstrate how DNA microarray experiments are performed. DNA microarrays, sometimes called DNA chips, reveal the expression of thousands of genes on a solid surface, such as a microscope slide. In this example, we’ll use yeast as a model system to illustrate one use of microarrays. One common use of microarrays is to determine
which genes are activated and which are repressed when two populations of cells are compared. Every gene is measured simultaneously. As an example, we’ll compare what happens
to yeast genes when cells are grown in aerobic versus anaerobic conditions. The cells grow and adjust which genes need
to be activated or repressed in order to survive. Now it is time to isolate the mRNA from both
populations of cells. The cells are spun in a centrifuge. Now that the cells have gathered in pellets,
we remove the liquid but not the cells. Next, it is time to extract the mRNA from
the cells. When we add the extraction buffer, the mRNA
is released into the solution. Next, we remove the RNA and place it in a
fresh tube. Now, let’s make the cDNA from the mRNA. Here we see three of the many mRNA molecules
from each tube of cells. Each mRNA is converted into red or green colored
cDNA. When the colored cDNA is made, the mRNA degrades. Then we combine the red and green cDNA, mixing
both colors into a single tube. At last, it’s time to look at the DNA microarray. In our experiment, a microarray or DNA chip
contains about six thousand spots, each spot is a different yeast coding sequence from
a different gene. Let’s choose three spots at random to follow
in detail. Each spot is made of DNA that can base pair
with its complementary cDNA. Here are partial sequences from each of the
three spots we are observing. Now let’s incubate the mixed cDNA with the
DNA chip. For the sake of our example, we’ll zoom
in and show that some of the labeled cDNA have bound to DNA in the spots and formed
base pairings. Here we see green and red cDNA bound to this
spot. Only red cDNA is bound to this spot. And only green cDNA bound to this other spot. In a real experiment, you would not see any
of this detail; you would only see the original microarray. Now we must wash off the unbound cDNA to see
what has bound to the microarray. Let’s detect the bound cDNA so it can be
visualized. We begin by placing the microscope slide containing
the microarray inside a scanner. We’ll examine the next phase of the process,
keeping our focus on the three spots we’ve been following. First, a green laser scans the microarray. The resulting image is stored on a computer
for later analysis. Now it’s time for the red laser. This image is also stored on a computer for
later analysis. Now we move to the analysis phase. After we eject and safely store the microscope
slide, we retrieve the red and green images from the computer and create a merged visualization. In the merged image we see an aerobic gene
labeled in green, an anaerobic gene labeled in red, and a gene labeled in yellow that
was expressed in both aerobic and anaerobic conditions. This is one example of how DNA microarrays
are used. In an actual experiment, quantitative analysis
would be conducted on all six thousand genes.

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