Download 3.1.4 Microarray

Activity 3.1.4 DNA Microarray
Introduction
We have approximately 20,000 to 25,000 genes. Traditionally, molecular biologists
could only study one gene at a time. Using this approach to investigate each
individual gene in the human genome would have taken a very long time. Advances
in technology now allow scientists to look at thousands of genes all at once using a
tool called a DNA microarray (also called a DNA chip or gene chip). Remember that
every cell in our body (with a few exceptions such as red blood cells) have copies of
each of our 20,000 genes. If all of the body’s cells have the exact same DNA, then
what makes a skin cell different from a nerve cell? The difference is in what genes
are turned “on” and “off” in each cell. In a skin cell, the genes for producing melanin,
a protein that gives your skin color, are turned on. In a nerve cell, the genes for
producing the neurotransmitter acetylcholine are turned on. The genes for producing
the hormone insulin are turned off in both skin and nerve cells.
DNA microarrays work by measuring the amount of mRNA for every gene that is
present in a cell sample so that scientists can determine which genes are turned on
and which are turned off. The reason scientists measure the amount of mRNA for
every gene is the basic assumption that if a gene is being transcribed to mRNA, it is
considered to be expressed. Scientists can then compare these results to those
found in another sample to determine the differences in gene expression between
the two samples.
How are DNA microarrays made? Using the information learned from the Human
Genome Project, scientists design primer pairs so that they can use PCR to make
copies of every gene in the human genome. They then separate the doublestranded DNA from each gene copy into single strands and place microscopic
droplets of each single-stranded DNA sample into ordered rows and columns on a
glass, plastic or silicon slide. This is called the DNA microarray. Each DNA
microarray can contain tens of thousands of genes.
Let’s take a look at cancer cells. Remember that cancer involves mutations with the
genes that regulate cell growth, division, and death. To be able to better diagnose,
understand, and treat cancer, it is important to understand what goes wrong with the
gene expression in cancer cells. DNA microarray technology allows scientists to use
the mRNA taken from two cells to determine which genes are turned on and which
genes are turned off. Scientists can therefore use DNA microarray technology to
help us learn about the differences in gene expression between a healthy cell and a
cancer cell. In a DNA microarray experiment, the two cell samples can either be
taken from the same patient or be taken from different patients, depending on the
purpose of the study.
In this activity, you will explore DNA microarray technology. As you learn about the
science behind this technology, you will perform a simulated DNA microarray to
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Medical Interventions Activity 3.1.4 DNA Microarray – Page 1
study the gene expression in a smoker’s versus a non-smoker’s lung cells and
analyze the DNA microarray results.
Equipment
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Computer with Internet access
Activity 3.1.4: Student Response Sheet
Laboratory journal
Carolina Biological DNA CHIPS: Genes to Disease Kit
o Glass slide
o Genes 1-6 Dropper Bottles
o cDNA Mixture Dropper Bottle
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Permanent marker
Colored pencils or markers
Micropipettor (20-200 µl)
Micropipettor (0.5-10 µl)
Disposable micropipette tips
70°C hot water bath
Digital camera (optional)
Safety goggles
Latex or nitrile exam gloves
Procedure
Part 1: DNA Microarray Virtual Lab
1. Obtain a Student Response Sheet from your teacher.
2. Go to the “DNA Microarray Virtual Lab” found at the University of Utah’s
Learn.Genetics: Genetic Science Learning Center’s website:
http://learn.genetics.utah.edu/content/labs/microarray/
3. Click on the picture of the microarray to begin.
4. Click on “Chapter 3: The Experiment!” to begin the virtual experiment.
5. Answer the questions on your Student Response Sheet as you work through the
virtual DNA microarray experiment.
6. Answer Conclusion questions 1 – 3.
Part II: Smoking and Lung Cancer
Grandpa Joe, Judy Smith’s father, has been a smoker for the past thirty years. Last
year, Grandpa Joe came down with a cold that turned into pneumonia. It took him more
than a month to recover. The family is very concerned he is going to develop lung
cancer. They heard about a study being conducted at the local hospital that is exploring
lung-cancer associated genes in smokers and non-smokers. The family convinces
Grandpa Joe to participate in the study in order to learn more about his risk for
developing lung cancer. The study is investigating six genes thought to be involved with
lung cancer using DNA microarray technology. The researchers hope to compare gene
expression of the six genes of interest between smokers and non-smokers in order to
gain more knowledge of what causes a normal lung cell to become cancerous. You
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Medical Interventions Activity 3.1.4 DNA Microarray – Page 2
have been assigned to the study. Your first task is to learn more about the six genes of
interest.
Below are descriptions for the six genes of interest:
Gene Name
Protein Function:
Prediction:
(and Symbol):
Gene 1: Human
This gene codes for a
carcinoembryonic
protein that is located in the
antigen
extracellular matrix. This
(CEACAM6)
protein is involved with
adhesion between cells
and is thought to be a
proto-oncogene and when
over-expressed is an
oncogene.
Gene 2: Surfactant protein
This gene codes for an
B (SFTPB)
extracellular protein. This
protein enhances the rate
of spreading and increases
the stability of pulmonary
surfactant, a lipid-rich
material that prevents lung
collapse by lowering
surface tension at the airliquid interface in the alveoli
of the lungs.
Gene 3: P53 tumor
This gene codes for a
suppressor (TP53) protein that is located in the
mitochondria and in the
nucleolus. This protein is
involved with cell cycle
checkpoints. This gene is a
tumor suppressor gene and
is thought to be the
“Guardian of the Genome.”
Gene 4: SRY
This gene codes for a
protein that is located in the
nucleus. The protein that
this gene codes for is
testis-determining factor
(TDF) which initiates male
sex determination. This
protein has no function in
lung cells.
Gene 5: Cytochrome P450
This gene codes for a
(CYP1A1)
protein that is located in the
endoplasmic reticulum. The
protein catalyzes reactions
involved in drug
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Medical Interventions Activity 3.1.4 DNA Microarray – Page 3
Gene 6:
Glypican 3(GPC3)
metabolism and
synthesizes cholesterol,
steroids, and other lipids.
The expression of this
protein is induced by some
polycyclic aromatic
hydrocarbons (PAHs),
some of which are found in
cigarette smoke.
This gene codes for a
protein that is located in the
plasma membrane and
extracellular matrix. The
gene controls cellular
response to damage and
may control cellular growth
regulation and apoptosis.
This gene is considered to
be a tumor suppressor
gene for lung cancer.
7. Highlight or underline any interesting or important information about the function
of each protein.
8. Predict how these genes will be expressed in a DNA microarray of a smoker
versus a non-smoker. Would you expect the genes to be induced in the smoker
(more expressed), suppressed in the smoker (less expressed), not expressed in
either the smoker or the non-smoker, or expressed the same in both the smoker
and non-smoker?
9. Record your predictions in the Prediction column of the above table.
Part III: Microarray Wet Lab
Now that you know more about the six genes of interest, your job is to perform a
simulated DNA microarray using tissue samples taken from Grandpa Joe and a nonsmoker’s tissue samples. The cDNA has already been prepared for you. You will first
prepare the simulated DNA microarray by spotting each of the six gene sequences onto
a glass slide. Actual DNA microarrays have thousands of microscopic DNA spots on the
slide. In this lab, our spots will be much larger than in a regular microarray, and you will
be able to view the spots without specialized equipment.
10. Put on safety gloves and goggles.
11. Use the permanent marker to number the six clear spots on the slide Genes 1-6.
Make sure not to touch the surface of your slide (handle it only by the edges).
12. Load 30 µl of Gene 1 onto the corresponding spot on your slide. You will need to
remove the top off the dropper bottle. Do this for each of the 6 genes. Use a
fresh tip for each gene. Your spots will harden in less than one minute.
o These spots represent the DNA sequences from six different genes.
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Medical Interventions Activity 3.1.4 DNA Microarray – Page 4
13. Draw a diagram of the slide in your laboratory journal. Make sure to clearly
indicate which gene is on which spot.
14. Obtain a cDNA dropper bottle (Hybridization Buffer) from your teacher and
carefully add 10 µl to each spot on your slide. Do not allow the micropipette tip to
touch the DNA spots.
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The cDNA dropper bottle contains a solution of labeled cDNA from Grandpa
Joe’s lung cells and a non-smoker’s lung cells mixed together. You cannot see
the color because the cDNA is very dilute. When added to the printed microarray
slide, the labeled cDNA in the solution will pair up with the complementary DNA
for each gene spotted onto the microarray, according to the base pair rules. As
each cDNA binds to the appropriate DNA spot on the slide, the labeled cDNA
becomes concentrated in that spot, allowing the spot to be visualized.
The cDNA used in an actual microarray is labeled with red and green fluorescent
dyes and the colors must be viewed using a fluorescent scanner to measure the
intensity of each spot. In this lab, the cDNA is labeled with pink (Grandpa Joe’s
lung cells) and blue (non-smoker’s lung cells) and do not need a fluorescent
scanner to view the results.
15. Place your DNA microarray slide onto a white piece of paper to observe results.
16. Draw your results under question 11 on the Student Response Sheet. Your
teacher may also take a photo of your slide. Include a description of the color of
each spot.
17. Answer questions 12 – 16 on the Student Response Sheet.
18. Wipe off the six spots on your slide with a paper towel. Wash and dry your slide.
You have just characterized the expression level of each gene from a smoker’s and
non-smoker’s tissue subjectively (i.e. deep blue versus light blue, deep pink versus light
pink, etc). When scientists analyze microarrays, they need to be able to quantitatively
measure the results. First scientists convert the colors to numbers according to the
intensity of red and green. For example, look at the following DNA microarray results for
four genes:
Gene A:
Gene B:
Gene C:
Gene D:
Red (Tumor
Cells)
Green
(Normal
Cells)
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Medical Interventions Activity 3.1.4 DNA Microarray – Page 5
Superimpose
d Image of
Green and
Red
Now we will convert these colors into numbers. The numbers represent the intensity of
the red or green color. The brighter the color, the larger the number to represent it. For
example, a value of 400 indicates a very bright light intensity, whereas, a value of 100 is
a dull color:
Gene A:
Gene B:
Gene C:
Gene D:
Red (Tumor
Cells)
Green
(Normal
Cells)
400
200
100
200
100
300
100
400
The next step is to calculate the ratio of red to green for each gene:
Gene A:
Gene B:
Gene C:
Ratio Red:
Green
(Tumor:
Normal)
Gene D:
400:100 =
4:1 =
200:300 =
2:3 =
100:100 =
1:1 =
200:400 =
2:4 =
4
0.67
1
0.5
The ratios can be used to give meaning to the results:
 When the ratio is greater than one, the gene is induced by tumor formation. This
means that the gene transcription was more active in cancer cells than in normal
cells.
 When the ratio is less than one, the gene is suppressed by tumor formation. This
means that the gene transcription was less active in cancer cells than in normal
cells.
 When the ratio is equal to one, the gene is not affected by tumor formation. This
means that the gene transcription was the same in cancer cells as it was in
normal cells.
 When the ratio is zero, the gene is not expressed in either cell.
19. Answer Conclusion question 4.
Now you will do this same process with the data you collected in your microarray. The
scale below represents the different shades you might see in your microarray. The
shades range from blue to pink as you go from left to right on the scale. Remember that
in our microarray, the cDNA from Grandpa Joe’s lung cells were labeled pink and the
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Medical Interventions Activity 3.1.4 DNA Microarray – Page 6
cDNA from the non-smoker’s lung cells were labeled blue. The numbers above the
scale represent the gene expression ratios that correspond with each color.
20. Match the colors in your microarray to those in the above scale. Your colors may
not be of the same intensity as those shown above. The colors you see will not
match exactly with the chart. Estimate the ratios as best you can, selecting ratios
between the given numbers whenever necessary.
21. Record your gene expression ratios for your microarray data under number 17 on
your Student Response Sheet.
22. Compare your results to your predictions. Were your predictions for each gene
correct? Explain your findings in your laboratory journal.
23. Answer the remaining Conclusion questions.
Conclusion
1. Imagine you want to learn more about Mike Smith’s osteosarcoma. How could
you use microarray technology to determine which genes have been affected in
his tumor cells?
2. What does it tell us if two genes show the same levels of expression in cancer
cells and normal cells?
3. What does it tell us if there are some genes that are highly expressed in normal
cells but not expressed in cancer cells?
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Medical Interventions Activity 3.1.4 DNA Microarray – Page 7
4. What range of ratios could indicate that a gene was not expressed in cancerous
tissue, but was expressed in healthy tissue?
5. When analyzing DNA microarray results, why are colors turned into ratios?
6. When analyzing DNA microarray results, what does a lack of color indicate?
(Note: in this experiment you saw a lack of color as white, but in real microarrays
it would be seen as black.)
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Medical Interventions Activity 3.1.4 DNA Microarray – Page 8
7. What further questions do you have about the gene expression differences
between Grandpa Joe’s tissue sample and the non-smoker’s tissue sample?
How might you design an experiment to answer one of your questions?
8. What can a DNA microarray teach us about oncogenes and tumor suppressor
genes?
9. Use the information learned from the DNA microarray to write an argument to
convince Grandpa Joe to stop smoking.
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Medical Interventions Activity 3.1.4 DNA Microarray – Page 9