Professor Dave again, let’s do some assays.
In biology, we study living organisms, and the key to understanding a living organism,
is to understand the expression of its genome.
What genes are present in the genome, and what proteins are produced when these genes
are expressed?
Beyond this, we may want to understand what is going on in a particular cell within an
organism.
How does a particular cell type perform gene expression?
How has gene expression been altered in a cancer cell?
A common approach for answering these questions involves identifying the mRNAs being made
by a particular cell during transcription.
From this information, a number of techniques can be performed, so let’s take a look at
these now.
As we said, analyzing gene expression will typically involve isolating mRNAs from a cell,
because from these we can perform the reverse transcriptase-polymerase chain reaction, or
RT-PCR.
This is where we take an mRNA and create a complementary DNA strand, in essence reverse
engineering the DNA template that would have generated the mRNA during transcription.
That is why the enzyme that performs this function is called reverse transcriptase,
as the process is essentially the reverse of transcription.
Whenever we do this, reverse transcriptase will use the mRNA as a template to generate
a single-stranded DNA molecule that is the complement of the mRNA.
As we recall, mRNAs have a poly-A tail, or a number of adenine RNA bases in a row, so
we can use a poly-dT primer, or a series of thymine DNA bases, to help reverse transcriptase
get going.
Once completed, another enzyme is used to degrade the mRNA, and then DNA polymerase
is used to synthesize the complementary DNA strand, resulting in double-stranded DNA,
which we will call complementary DNA, or cDNA.
Now we can examine some applications of this approach.
Say we want to know precisely when a gene is being expressed during the embryonic development
of an organism.
We could isolate the mRNAs from different stages of development, and perform RT-PCR
to get cDNA fragments that correspond to all of these mRNAs.
Then we could amplify a specific cDNA molecule that represents the gene of interest, using
the polymerase chain reaction, by employing a primer that is specific to the gene of interest,
so that only the cDNA representing the gene of interest is amplified.
We can repeat this process for each of the stages of embryonic development we are testing.
Then we can perform gel electrophoresis, with a column for each stage of development, whereby
only the amplified product will be relevant, since it will be so much more abundant than
anything else.
If we get a lot of the gene of interest, it means that the mRNA it produces was present
in the sample during that stage, because its presence was necessary in order to get the
corresponding cDNA in the first place.
So this is how we can tell when a particular gene is being expressed in the embryo.
Quite similarly, this approach can be used to determine which tissues within an organism
are expressing a particular gene, by extracting mRNA from different tissues, and going through
the same process with cDNA and amplification to see which tissue samples contained the
mRNA associated with the gene of interest.
But more importantly, biologists are often interested in understanding genome-wide expression.
In other words, we want to understand the ways that many different genes act together
to produce and maintain a functioning organism.
To do this, we will typically perform something called a DNA microarray assay.
This is a powerful technique involving a huge grid of tiny spots, and in each well sits
many copies of a single-stranded DNA fragment called a probe, attached to a solid surface,
which each represent a particular gene.
So we can think of this as a grid of many different genes of an organism, or even all
of them, ideally.
Then, by the process we already discussed, mRNAs that are made in a cell of interest
are isolated, and reverse transcription is performed, to generate cDNAs.
These cDNAs are labeled with fluorescent tags, and introduced to the array, allowing them
to hybridize with any complementary DNA sequence they can find.
Now recall that these cDNAs will have sequences that should be identical to segments of the
genes that produced the mRNAs, because DNA is the template for the mRNA during transcription,
and the mRNA is the template for the cDNA during reverse transcription.
So one of the two strands of some cDNA molecule made from an mRNA ought to bind to the DNA
fragment in the particular well that can produce that mRNA in the first place, since they will
be highly complementary, and once time is given for hybridization to take place, any
cDNA that exhibits little to no binding is washed away.
Wherever binding has occurred, this is indicated by the fluorescence of the cDNA, and is clearly visible.
If we label different samples with different colors, such as different tissue samples,
we can then get an enormous amount of qualitative data all at once.
Everywhere we see one color, such as red, red cDNAs are bound, meaning that particular
gene is expressed in the tissue that we labeled red.
Where we see green, that gene is expressed in the tissue that we labeled green.
If we see yellow, that means both the red and green cDNAs are binding, so we see the
intermediate yellow color, indicating that the gene is being expressed in both tissues.
And if we see little to no coloring, the gene is not expressed in either tissue, and no
binding will occur.
This technique is immensely useful in a variety of contexts.
Say we want to know how gene expression has been altered in a cancer cell.
We can compare the mRNAs in a regular cell and a cancer cell on the same array, and as
simply as identifying a color on a grid, we can know precisely which gene has been altered.
Then we can just sequence that gene and know precisely where mutation has occurred, and
therefore get information about how the resulting protein has been altered, which allows us
to understand on a fundamental level why the cancer is occurring, and suggest new approaches
for treatment.
There are at least a dozen other common applications of this technique as well, which have a range
of utilities.
All of this makes the DNA microarray assay an indispensable tool in the molecular biology laboratory.