Download Student Background: How A Microarray Works

Student Background: Molecular Biology in Relationship to DNA Microarray Technology
In your study of biology, you have learned that your cells contain DNA. DNA is really just a very
detailed set of instructions on how to make an organism. Your DNA is the set of instructions that
makes you, YOU. Think of DNA as a blueprint, just like the ones construction workers use to
construct buildings. The blueprint is essential to construction, but you can’t sit or eat or learn in
a blueprint, you need to build the building for that. That’s where RNA and proteins come in.
There’s a saying in molecular biology and it goes “DNA RNA protein.” This statement is
sometimes called the central dogma of molecular biology and it describes the flow of biological
information. DNA stays in the nucleus of the cell where it is protected by the double membrane
called the nuclear envelope. Only certain substances are allowed to get into the nucleus, and
those special proteins have a sequence called the NLS (nuclear localization signal) that allows
them to get in! It’s like having the secret code to get into a safe. No code, no entry! Your DNA is
protected just like a safe, because the DNA is so important.
RNA is a copy of the DNA, but it’s not a copy of the whole thing. RNA is a copy of a very
specific sequence of DNA, a specific set of instructions on how to make one specific protein.
Let’s say something happened to your classroom in your building during the night and when you
got to class today, the whole room had to be rebuilt all the way down to the wiring and plumbing.
You need to go back to the original blueprints to figure out how to rebuild your classroom. You
don’t need the blueprints for the whole building though, just the ones for your classroom, so you
make a copy of your classroom’s blueprint. DNA is the blueprint, it stays in the “storeroom,” the
nucleus. The RNA is similar to the storeroom copy, and it leaves the nucleus. The process of
making the DNA to RNA copy is called transcription. After the RNA leaves the nucleus it heads
to cellular structures called ribosomes. The ribosomes build the proteins based on the
information contained in the RNA, as such; they are essentially your cellular construction
workers. This process is called transcription. It is the proteins that carry out the functions of the
cells; it is proteins that allow red blood cells to carry oxygen, skin cells to be elastic and muscle
cells to contract. The DNA itself can’t do those things; the proteins do the work of the cells.
All of the cells of an organism contain the entire DNA and therefore have all the information on
how to make a whole other you. But, think about it, do your skin cells need to make insulin? No,
that’s the job of islet cells in the pancreas, and those pancreatic cells don’t need to make an
antibody, that is the job of your immune cells. Different cells have different functions and make
different proteins. So even though all the cells have the DNA sequences, called genes, the
same genes are not active in each and every cell. If a gene is expressed, that means it is turned
“on,” or active. The genes expressed by a cell at any given time will dictate what the cell is
doing. Remember the central dogma of molecular biology which is DNARNAprotein. If a
gene is expressed, that means the DNA is transcribed into RNA and the protein is made, the
gene is “on.” If the gene is not expressed it stays encoded in the DNA, but the protein isn’t made
and the gene is “off.” Different cells turn their genes on and off in response to various signals
and stressors in the environment. Studying which genes are “on” or “off” in a cell can help
scientists determine why a cell behaves the way it does. For example, normal cells are under
the regulation of the cell cycle, and they only divide when they are “told” to by cellular signals.
Cancer cells have escaped this regulation and they divide even if they aren’t “told” to. Masses of
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these out of control cells grow into tumors. Scientists have discovered that one of the
differences between normal cells and cancer cells is which genes are turned “on.” DNA
microarray technology is a way to study which genes are active in a cell. In cancer biology,
microarrays have been widely used to look at varying gene expression between normal and
malignant cells. In this activity you’ll be learning how DNA microarray technology works.
How a DNA microarray works:
DNA microarray technology is fairly simple to understand once you realize that this technique
exploits the same property of complementary base pair binding that holds double stranded DNA
together. DNA microarray technology takes advantage of the hybridization of complementary
strands of nucleic acids. This means that DNA or RNA strands that complement each other will
stick together. Remember that the adenine is complementary to thymine in DNA and uracil in
RNA, and guanine is complementary to cytosine.
There are two components to the DNA microarray: 1) the DNA microarray itself and 2) the
sample(s) being examined. Hybridization that occurs between the DNA on the microarray and
the mRNA from the sample can illustrate what genes are active in a cell at a given time.
The microarray:
The actual microarray itself is a solid surface usually glass, plastic or silicon
that has been spotted with thousands of short segments of DNA called probes,
and they are so small you can hold them in the palm of your hand. The DNA
probes are portions of genes that the scientist is interested in. DNA
microarrays are manufactured for scientists by corporations. Check out this link
(http://www.affymetrix.com/products_services/arrays/index.affx) to see all the
different kinds of microarrays available!
Here’s a simple illustration showing a DNA microarray that has three probes:
#1, #2 and #3.
1
2
3
TAGC
CTTG
ATGC
The samples:
The samples are the mRNAs extracted from the cells you are interested in. Remember that if a
gene is “on”, mRNA will be in the cell. The first step will be to extract the mRNA from the cells of
interest. Of course this will mean opening the cell membrane and getting the cell to spill its guts.
Then, you have to isolate just the RNA from the cell guts. Using a series of chemicals and
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washes, this is really pretty easy. The only problem is that mRNA is single stranded and is very
fragile. Inside the cell mRNAs degrade shortly after translation, and that’s a good thing. If the
mRNA hung around too long, it might get translated again and again and again and the cell
would end up producing much more protein than was intended. In the laboratory, mRNAs are
also very vulnerable to degradation by the same RNAse enzymes that breakdown mRNAs
inside the cell. So, in order to preserve the stability of the sample, mRNAs are turned back into
DNA through a process called reverse transcription. The DNA that is a copy of the mRNA is
called cDNA (copy DNA). It is the cDNA that is used in the microarray technique. Reverse
transcription is transcription, in reverse. So instead of converting DNARNA, reverse
transcription converts RNA DNA. A specific enzyme called reverse transcriptase can convert
single stranded vulnerable mRNA into single stranded, but much more stable, cDNA. Reverse
transcriptase reads the mRNA and uses complementary base pairing to make the cDNA.
Remember complementary base pairing would be:
mRNA
A
U
G
C
cDNA
T
A
C
G
During this process, the cDNA is also being labeled. This means that one of the nucleotides,
let’s say A, has been radioactively or fluorescently labeled so that you can “see” it later. Let’s
walk through a process so you can see complementary base pairing at work.
We will begin with mRNA with the sequence:
UUAGGUAACCUUGUAAU
The mRNA is isolated from the cell, and in the laboratory is reverse transcribed into cDNA with
a labeled A* nucleotide that makes it appear GREEN.
A*A*TCCATTGGA*A*CA*TTA*
The labeled cDNA is now your sample.
Putting the DNA microarray and the sample together:
Now it’s just a matter of getting the cDNA onto the microarray to see where your sample sticks!
First, you put the cDNA onto the microarray and then wash it off. The cDNA that “found” its
complementary sequence on the microarray will bind with high-affinity and it won’t come off in
the washing process. cDNAs that didn’t find their complement will wash right off.
Can you predict what sequences would be necessary to hybridize to our DNA microarray from
the previous page? Remember that complementary sequences will bind. (Answers are on the
next page in blue.)
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1
2
3
TAGC
CTTG
ATGC
ATCG
GAAC
TACG
Now, let’s look at our sample sequence again to see if our sample would have stuck to the chip.
A*A*TCCATTGGA*A*CA*TTA*
Can you predict if and where our sample would stick? It won’t stick to probe #1 because the
sequence ATCG isn’t in our sample. It won’t stick to probe #3 because the sequence TACG isn’t
in our sample. But, it will stick to probe #2 because GAAC is in our sample!
A*A*TCCATTGGA*A*CA*TTA*
So you can imagine that the DNA chip looks like this: there is hybridization between probe #2
and our sample, so our sample is stuck to the chip!
1
2
3
TAGC
CTTG
ATGC
Seeing the results:
A*A*TCCATTGGA*A*CA*TTA*
Of course, you can’t see the probes or the cDNA with your eyes, but, remember when we
reverse transcribed our mRNA into cDNA we added that green labeled A. That label will allow
us to visualize which probes have hybridized with our sample. Our label was green, which
means that means any place on the microarray that glows green has labeled cDNA hybridized
to it.
1
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2
3
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Interpreting the results:
Now we’ve got a glowing green spot on a plate, but what does it mean? Of course it means that
our sample is stuck there. But what does THAT mean? Remember that the probes on the chip
are portions of genes that the scientist is interested in, so in our case, it means that our sample
had mRNA for the gene #2. It means that in our sample, gene #2 was being expressed, but
genes #1 and #3 were not. Now we know more about what was going on in our cell.
A real microarray is a bit more complicated: (you knew that was coming!)
Now you understand how cDNAs hybridize to their complementary DNA probes that are
encoded in the microarray. In our example above, we had just one gene expressed, but
remember that at any given time, a cell will have multiple genes being expressed, not just one.
So, when you isolate your sample, there may be many genes that are “on.” Let’s look at a larger
microarray example with five different genes probes.
1
2
3
4
5
These results show that our sample is expressing genes #2, 4 and 5. In a real microarray, you
can examine the expression of thousands of genes.
What if we want to look at more than just one sample? What if we want to compare gene
expression from two different cells? You can run both samples on the same microarray AS
LONG AS, when we reverse transcribe the second sample, we label the cDNA with a different
color. We will use red for our example. Let’s look at the results when we apply two differently
labeled cDNA samples to the same microarray.
1
2
3
4
5
We already knew that sample A was expressing gene #2, 4 and 5. What do the new results tell
us? Since sample B was labeled red we know that sample B is expressing genes #1 and #3.
What about the yellow disc? Where did that come from? In a microarray, the probe is yellow
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when both the green and red labeled cDNAs are hybridized to the same probe. Review the
results:
Gene
Sample A
Sample B
1
No
Yes
2
Yes
Yes
3
No
Yes
4
Yes
No
5
Yes
No
You can see that there is different gene expression in our two samples. Imagine the possibilities
for DNA microarrays! DNA microarray technology is revolutionizing the way we look at DNA.
DNA chips with thousands of gene probes are available for many different species and allow
scientists to get genetic information FAST. DNA microarrays are used for clinically for disease
diagnosis, genotyping and of course in basic research.
Online Resource Microarray animation and background:
http://www.bio.davidson.edu/Courses/genomics/chip/chip.html
http://science.education.nih.gov/newsnapshots/TOC_Chips/Chips_RITN/chips_ritn.html
Activity 1: DNA Microarray Background
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