It is said that knowledge is power and with
biotechnology we can see in this presentation how it is
true where the knowledge about molecular
biological processes has been
brought to develop powerful
I am going to talk about three of those
techniques in this presentation.
DNA amplification, microarray
analysis and 2D gel analysis.
Now, knowledge about the process of
DNA amplification has been applied
in a technique called the polymerized chain reaction
that has revolutionized the way that we
see and analyze DNA.
The polymerized chain reaction allows a person
to amplify specific DNA segments out
of a larger DNA millions of times.
It's very popular for a forensic analysis
and if you ever watched a crime
show on TV you have probably seen
PCR being applied to action.
The process of PCR or polymerized chain reaction
is derived simply by copying some
ideas from cellular DNA replication
and then using this in a noble way to
accomplish the replication of DNA
that one desires only
to copy in a specific way.
The advantage of PCR is that it is
a very simple technique to perform
A high school student can be
trained to do PCR in about an hour.
It requires sequence knowledge to start and that's
one of the primary requirements of PCR, as we shall see.
Now the PCR technique uses
the knowledge of the sequence
to design and chemically synthesize
DNA primers flanking the region to be
amplified. Now I need to explain that.
In a previous lecture I talked
about how DNA polymerase uses a primer
to start DNA replication and
in the cell that primer is RNA.
Now the problem with the RNA
primer is, it’s got to be removed
and then replaced by something else.
That's kind of a complicated process.
If we wanna replicate DNA in a test
tube and we wanna do it efficiently,
We start with a DNA primer.
And the beauty of a DNA primer is that it
defines a starting point for replication.
As since a primer has a specific sequence
one can design that sequence and then allow it to
find the right place to form base pairs and that will
define where the replication is occurring, as we shall see.
To bring this process about, we have to take a
target DNA. So this DNA might be let's say a human DNA
and this human DNA contains a region
that we are interested in analyzing.
Maybe it's a genetic mutation that a person has
suffered that we are interested in determining what it is.
In each case we would know the sequences around the
DNA that we are interested in studying, so, let's says,
for example, we have a person who has sickle cell anemia.
And sickle cell anemia involves a
mutation within hemoglobin gene.
We know the sequences of the hemoglobin gene but we don't
know the sequences of the mutation in the middle of the gene.
So we could design DNA primers that are
complementary to either end of the hemoglobin gene,
make those and make them so that they will now
form base pairs with the end of the hemoglobin gene within the DNA.
We take that target DNA which is
the person who has the mutation
we take their DNA, mix it with the primers,
mix it with a 4 dNTPs and here is the kicker,
a thermostable DNA polymerase. Now we will see in
a minute why that thermostable DNA polymerase
is important. The thermostable
part means that it's resistant
to denaturation by heat. Most enzymes fall
apart in heat, a thermostable one doesn't.
We take this whole system. This mixture that
I have just defined to you and we add it
to something called a thermocycling system.
And what a thermocycling system is, it is a device
that will heat up this sample and then cool
it to various temperatures as we shall see.
And so this begins a process that
I am going to describe next.
So we can see for example here the
target DNA of the person that has
the mutated hemoglobin gene on top.
Its a duplex DNA and I
have made it short here.
But the reality is that we would have the entirety of that
person's chromosomes. So there is a lot of sequences here.
The first step in the process of employing
PCR is to pull the strands apart.
Now this kind of mimics what the helicase was doing in DNA
replication expect for here we are physically pulling them apart.
But how to do pull them apart? The way that you pull
them part that you heat the mixture to near boiling.
When you do that, the hydrogen bonds
that hold the DNA duplex together
are broken and the strands completely come
apart. So the strands separate here.
The second part is to cool that temperature down
so that the primers that were mixed in the solution
can form base pairs with their appropriate sequence.
Now if you make the primers of the right length,
the only place is where they will form the base pairs
are where you intended them to, on either
side in this case of the hemoglobin gene.
This process requires a specific
temperature called an annealing temperature.
So the thermocycler heated
the system up to boiling
and now it's cooled down to this magical temperature where
the primers will form base pairs with their complements.
The third step then is for the DNA
polymerase to replicate those strands.
Well, the DNA polymerase
was already in the mixture
and remember we used a thermostable
DNA polymerase and because of that
it didn't get damaged by
that boiling that we did.
The DNA polymerase uses
the 4 dNTPs to replicate
the strand of interest and we can see the
replication now that is occurring here.
So after that's happened the primers defined
the ends. So I see one end in yellow
and I see another end in green.
This replication proceeds and
now what happens is the primers
direct replication over and over
and over of the same strands.
So notice that the two strands that we're copying
are just the top and the bottom strand. They are
actually the same sequence.
So then we get a duplex
that's made from that.
It means that we started
with one duplex in step 1.
But by step 3 we have two
copies of that same duplex.
But you can start to see what happens here.
Every time we do a cycle of boiling,
annealing and replicating, we
double the number of strands.
Now that might not seem like a big deal. But if
you do 30 times you have 2 to the 30th
more strands than you started with at
least in theory. That's over a billion.
So that means you can take very tiny amounts
of DNA and make incredible quantities out of it
using this method. This is why it is
a very powerful tool in a crime scene.
It doesn't take very much of a suspect's
DNA to get this kind of analysis done.
This process is typically repeated for 30 to 40 cycles although
there are some processes where it's done even more.
Now this powerful technique
is one of many that we use
to analyze sequences. So for example
analyzing DNA is done by PCR
but there are other types of analyses that we are
interested in doing. And these types of analyses
I'd like to sort of lump together
and call Omics analysis.
We will see how that comes into play in just a moment.
The technological advances really are what
have made the Omics analysis possible.
Omics methodologies focus on individual molecules
within a cell but across a broad spectrum.
So what happens for example that if we talk
about genomics, there is one of the Omics,
we are studying every
DNA sequence in a cell.
Now 30 years ago that was inconceivable,
nobody knew how to do that
now we can sequence a genome in
very short periods of time, a week.
We can do transcriptomics in which we are
analyzing all of the transcripts of a cell.
Now, the transcripts of course are the RNAs
that are being transcribed for making protein.
If we know all of the RNAs of a cell
and how many of each is being made
we have an amazingly broad piece of information
about what the cell is doing and
how much of it it's trying to make.
Proteomics is another one of the Omic that's
involved in the study of protein expression.
So just like we can use transcriptomics to tell
us how many and what kinds of RNAs are being made,
proteomics allows us to determine how many and
how much of each protein is being made in a cell.
So metabolomomics is an
analysis of the metabolome,
a metabolome of course corresponds to all of
the metabolites that are being made in a cell.
That could include all of the molecules
made in a citric acid cycle
and what quantity of each is being made.
All of the molecules being made in glycolysis
and what the quantities of each of those are.
In other words, all of the different
biochemistry that is happening
spectrally across the entire cell.
Now that's the advantage of
Omics type analysis and there are dozens of
Omics disciplines now that people have
developed to do these kinds of analyses.
And as a result of this we are actually
able to understand at a system level
what's happening in the cell. Instead
of understanding at an individual
molecular level what's happening in the cell
and what happens with that system analysis
allows us to better understand
what life is really all about.