MICROARRAY ACTIVITY
A copied and modified activity originally by Betsy Barnard
www.nabt.org/sub/pdf/068-03-0002.pdf
Name: __________________________________
Background
A DNA microarray (DNA chip) is an ordered array of different known sequences of DNA
(~20-70 bases long). These DNA sequences represent many of the genes in an
organism. Many copies of each different sequence are stuck to one “spot” on a solid
surface (glass). A DNA chip can have thousands of different spots, representing
thousands of different genes. A microarray can have tens of thousands of genes in an
area smaller than the size of a postage stamp.
In this activity, each DNA sequence stuck to the chip is called a probe. DNA chip
technology is based on the basic chemistry of DNA. Adenine pairs with thymine and
guanine pairs with cytosine. This base complementarity is what allows DNA from cells
to bind specifically to known DNA sequences (probes) on a chip. Since a cell expresses
hundreds or even thousands of genes at any given time, a “snapshot” of gene activity
can be inferred using microarray technology. Without this technology, it could take years
to analyze each gene one at a time.
In this activity you will use only a few probes, rather than thousands, to detect
differences in gene expression between seedlings that have been sprouted under two
different conditions: light and dark.
A real microarray experiment requires a high level of skill to prepare the samples and
sophisticated equipment and software to collect and analyze the data. Because this
technology has only been commonly available since the 1990s, it is still being
developed, refined, and perfected. One thing is certain: This technology is not going
away—in fact, it is being adapted for studying other types of molecules in cells such as
proteins and carbohydrates.
Materials
American Scientist Article on Microarrays
Scissors
Masking Tape
Red, Green, and Yellow colored pencils
Red and Green paper for cDNA sheets
Procedure
This simple microarray experiment involves looking for differences in gene expression
of seedlings grown under two conditions: One set of seedlings receives full light and the
other set germinates completely in the dark. Light-grown seedlings are green with open
cotyledons (first leaves) and short hypocotyls (the stem between the root and the
cotyledons). Dark-grown seedlings are yellowish with closed cotyledons and long
hypocotyls.
1. Observe the mRNA transcripts expressed in the seedlings grown in light conditions
(the red paper handout). Each transcript represents a gene that was expressed in
that organism at the time the seedlings were harvested. Note: some transcripts are
rare, some are abundant (abundant means many copies are present). Transcripts
may also vary in length, since DNA coding regions vary in length. (In reality, mRNAs
are typically much longer than those used here.)
2. For each mRNA transcript imagine that you are the enzyme “reverse transcriptase”,
which transcribe backwards from RNA to DNA, and determine the complimentary
DNA, cDNA, strand that would be created. Write the appropriate DNA nucleotides
abbreviations in the small rectangle labeled cDNA, and then use scissors to cut out
each cDNA rectangle. The cDNA rectangles were copied onto red paper to
represent the red fluorescent tag that is commonly added to the control cell
condition. In this case the seedlings grown in the light conditions.
3. For each cut out cDNA, find the appropriate complimentary DNA spot on the DNA
microchip that has the complimentary DNA nucleotides sequence, and tape that
cDNA rectangle in the space under the DNA probe sequence (make sure to leave
room to tape other such cDNA’s under the same probe sequence). This set
represents DNA hybridization. Be sure to obey the rules of base complementarity,
and remember that double stranded DNA is antiparallel. For this activity you may
assume the temperature is high enough so that there must be at least four bases in
a row that match, otherwise the cDNA won’t bind to the probe DNA. (In reality,
microarrays use DNA probes that are 20-70 bases long and cDNA that is much
longer than 6 bases. Researchers use sophisticated computational methods for
determining how many bases must match for binding to be specific to a single cDNA
sequence.) Assume that you have washed away the non-complimentary transcripts.
4. Now, complete steps 1 through 3 for the mRNA transcripts expressed in the
seedlings grown in the dark conditions (the green paper handout).
5. Next, for each DNA probe or gene, note whether the probe DNA hybridized with only
the light cDNA, only the dark cDNA, both cDNA types, or neither. If only the light
cDNA hybridized color the circle to the right red. If only the dark cDNA hydridized
color the circle to the right green. If both types of cDNA hybridized color the circle to
the right yellow, and if neither did so leave the circle colorless. When finished
answer the questions that follow.
Questions
1. Identify which genes are expressed in the control (light condition) and experimental
(dark condition) groups by looking at the DNA microchip. To demonstrate your
understanding, fill out the following Venn diagram with the gene functions. Note
those genes that neither express below the Venn diagram.
Light Condition
Both
Dark Condition
2. Summarize the results for light condition and make conclusions describing if the
genes expressed in this condition are sensible. Make sure to explain why some
transcripts are more abundant than others.
3. Summarize the results for dark condition and make conclusions describing if the
genes expressed in this condition are sensible.
4. Explain why the some genes were not expressed in either condition.
5. What was the outcome of each control spot, and what does this indicate?
6. Are there any cDNA target molecules (from mRNA transcripts) that did not match the
probe spots on the DNA chip? What does this indicate?
7. Which steps in the following flowchart correspond to the steps you completed?
Explain how you modeled those steps.
Homework Questions
Read the article “Gene Chips & Functional Genomics” to answer the following questions.
1. What is the next most important thing now that we have sequenced our own
genome?
2. What invention has recently been developed? Name the three industries that merged
in the development of this invention?
3. Although all cells have the same DNA, why are specialized cells of the muscle, skin,
nervous system, and immune system different?
4. Give an example of the new functional genomics.
5. Describe how Patrick Brown at Stanford used knowledge of the following
technologies to develop microarrays.
a. Semiconductor Technology
b. Laser Technology
c. Mathematics and Computer Technology
6. How can different levels of gene expression be determine using microarrays?
7. What is the new field of bioinformatics?
8. How can microarray technology benefit the study of toxicology and pharmaceuticals?
9. If you were an expert in this field what do you think would be an interesting question
to address using microarray technology?
DNA Microarray
5’ T C A C 3’
5’ A C C C 3’
gene 1 – Abscission (loosing leaves)
gene 9 – Reproduction (makes pollen)
5’ A T T A 3’
5’ C G G G 3’
gene 2 – Cell Elongation I
gene 10 – Cellular Metabolism
5’ A A C C 3’
5’ A T G G 3’
gene 3 – Light Signal Processor I
gene 11 – Yellow Pigment
5’ A T T T 3’
5’ A T A T 3’
gene 4 – Light Signal Processor II
gene 12 – Reproduction (makes eggs)
5’ C T C C 3’
5’ G T C A 3’
gene 5 – Stress Response
gene 13 – Salt Response
5’ G A C C 3’
5’ A A G G 3’
gene 6 – Cell Division (Mitosis)
gene 14 – Chlorophyll Pigment
5’ C A T A 3’
5’ T C G G 3’
gene 7 – Temperature Response
negative control – Animal Gene
5’ A G T A 3’
5’ T T G G 3’
gene 8 – Cell Elongation II
positive control – Water Transport
Light Grown mRNA
light conditions
mRNA: 5’ U U G G C A 3’
cDNA:
light conditions
mRNA: 5’ A A G G C C 3’
5’
cDNA:
light conditions
mRNA: 5’ A A G G C C 3’
cDNA:
light conditions
mRNA: 5’ A A A G G C C 3’
5’
cDNA:
light conditions
mRNA: 5’ C G G G A U 3’
cDNA:
5’
light conditions
mRNA: 5’ A A C C G U 3’
5’
cDNA:
light conditions
mRNA: 5’ A A A U U U 3’
cDNA:
5’
5’
light conditions
mRNA: 5’ G A C C U 3’
5’
cDNA:
5’
Dark Grown mRNA
dark conditions
mRNA: 5’ U U G G C A 3’
mRNA: 5’ C G G G A U 3’
5’
cDNA:
dark conditions
cDNA:
dark conditions
mRNA: 5’ A U G G G C 3’
cDNA:
dark conditions
mRNA: 5’ G A C C U 3’
5’
mRNA: 5’ C U C C U A 3’
dark conditions
mRNA: 5’ C U C A A 3’
5’
cDNA:
dark conditions
mRNA: 5’ A U U A G C 3’
cDNA:
5’
cDNA:
dark conditions
cDNA:
5’
5’
dark conditions
mRNA: 5’ A G U A C 3’
5’
cDNA:
5’