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- Welcome back to our Experiment Explained Series.
The goal of this video series is to provide you with short,
digestible, and high-yield reviews
of experimental techniques that you're likely
to see on the MCAT.
My name is Vikram Shaw, and today we'll be looking
at everything you need to know
about Polymerase Chain Reaction or PCR for the MCAT.
We'll look at the figures, controls and common places
you'll see PCR on the biology
and biochemistry section of your exam.
By the end of this video, you'll feel confident
in your ability to analyze PCR experiments
that may show up with any context on the MCAT.
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PCR is a common technique used to make copies
of a specific nucleotide sequence.
To understand how these copies of nucleotides are made,
let's first review the process
of how a cell duplicates its own DNA.
DNA duplication happens every time a cell divides.
For a cell to make a copy of its DNA genome,
a few enzymes are required.
Let's take a look at this image.
Enzymes called helicases and DNA topoisomerase
are used to unzip the two strands of DNA
that are bonded with each other.
These two strands need to be separated,
so that the other enzymes have access
to the nucleotides in each strand,.
An enzyme called DNA polymerase
add short fragments of nucleotides
to a single-stranded piece of DNA.
These short fragments are called primers,
and are the starting point for the formation
of a new copied strand of DNA.
These primers are absolutely necessary
for DNA to be copied.
Without the presence of primers,
the next enzyme DNA polymerase won't be able to function.
DNA polymerase is the workhorse of DNA replication.
It's a huge enzyme that continuously adds new nucleotides
to the growing chain of DNA.
The end product is a long continuous strand
of newly-synthesized DNA that's complimentary
to the original strand of DNA.
Since DNA is a double-stranded molecule,
and since both strands of DNA
can be replicated at the same time,
the end result after one round of replication
is four total strands of DNA.
After the second round of replication,
there are eight strands of DNA.
And after the third round of replication,
there are 16 strands of DNA.
So what does DNA replication had to do with PCR?
Well, if you understand all of the elements
of DNA replication, then you're very well prepared
to understand PCR.
That's because PCR is essentially the same process,
but adapted to happen in test tubes instead of cells.
Suppose we have a strand of DNA
that needs to be duplicated in a PCR environment.
The first step in duplicating DNA
is to untwist the double helix,
and separate the two strands.
In the cell, the enzymes helicases
and DNA topoisomerase took care of that.
In PCR, we can do this by applying a little bit
of heat or temperature.
This process of adding heat to split the DNA
into two strands has a special name,
which is known as denaturing.
The second step of DNA synthesis was adding some primaries
to both of the DNA strands.
Remember, that a primer is just a short strip
of single-stranded nucleotides.
In the cell primers are made
by an enzyme called DNA primase.
In PCR, these primers can be synthetically made
according to a sequence the researchers desire,
and then are simply added to the test tube.
The process of adding single-stranded primers
and allowing them to bind is called annealing.
The third step of DNA synthesis
was to continuously add a new nucleotides
to the growing strand of copied DNA.
In the cell, this was done
by an enzyme called DNA polymerase.
In PCR, we simply use a different form
of this enzyme known as taq polymerase.
Taq polymerase is a heat-stable polymerase,
which means it won't denature at the high temperatures
we need in the PCR reaction.
The process of growing the complimentary strands
is known as elongation.
So to recap, there are three distinct steps
in the PCR process.
Step one, denaturation.
The double-stranded DNA in the test tube is heated
to slightly higher temperatures
in order to melt and separate the two strands.
Step two, annealing.
Specific primers are added to the test tube,
and the test tube is cooled to allow the primers
to bind to single-stranded DNA.
And step three, elongation or extension.
The enzyme DNA polymerase is used to make a long
and continuous strand of DNA.
After these three steps have been completed,
the end result is two identical copies
of the same DNA sequence.
The three steps can then be done again,
and then result would be four identical copies
of the same DNA sequence.
So let's take a moment and step back.
We haven't yet defined the phrase PCR.
What do these letters stand for?
Well, they stand for the phrase Polymerase Chain Reaction.
Let's break that down a little bit more.
The P in PCR stands for polymerase.
Remember that DNA preliminaries is the workhorse enzyme
that's used to put a long continuous string
of nucleotides together.
The C and the R and PCR stand for chain reaction.
Once the first round of DNA duplication is done,
the new copies of DNA that were made
can actually be used these templates
for the next round of duplication.
So as long as there are enough starting materials,
the process of DNA duplication and PCR
can repeat itself over and over and over again
without running out of DNA templates.
So this is what's typically done during PCR experiments,
a machine known as a thermocycler simply repeats
the three steps over and over again.
A typical PCR experiment or protocol might result
in 30 to 40 rounds of duplication
that can be completed in just a few hours.
After those 30 to 40 rounds of duplication,
there could be as many as 2 to the power of 30,
or 2 to the power of 40 copies of DNA,
which has millions and millions of copies of DNA
that were created
from just one original double-stranded piece.
So to summarize, PCR is a laboratory technique
that can be used to copy pieces of double-stranded DNA.
It can be truly useful in amplifying DNA,
or taking a really small amount of DNA
that might be hard to detect,
And making a large number of copies
that would make it much easier to detect.
For polymerase chain reaction on the MCAT,
there are a few key ingredients
you're gonna need to know.
So here's what we need.
The original piece of double-stranded DNA
to be amplified.
Primers, which are specifically designed
by the experiment and synthetically made.
DNA or taq polymerase, dNTPs, which are free nucleotides
that can be incorporated by DNA polymerase,
and buffers, which maintain the environment
of the solution, so that the DNA polymerase
can work optimally.
There are a few variations on PCR that you might see
on the biology and biochemistry section of your MCAT,
qPCR or quantitative PCR is used to quantify the amount
of DNA that has been produced.
So this might be determined using a fluorescent probe
that can be visually seen when it's bound
to double-stranded DNA.
Reverse transcriptase quantitative PCR
is used to amplify pieces of RNA.
To do this, a reverse transcriptase enzyme is first used
to convert the RNA to its complementary DNA.
That's what the RT stands for.
This first step needs to be done,
because DNA polymerase can only work
with DNA and not RNA.
So researchers often use RT-qPCR
again real-time quantitative PCR to compare different levels
of mRNA present in a cellular sample.
For example, they may look at tumor expression
of an mRNA molecule before and after treatment
with a potential new therapeutic.
Once the PCR is completed, the large amount of DNA
that's made can be subjected to other experiments,
such as Southern blot.
To review what a Southern blot is,
you can check out our last Experiment Explained video,
discussing Western blots and related techniques.
Now, that you have a good understanding of PCR,
let's take a look at some real-world examples
of applications you might actually see on the MCAT.
Here's an example of a PCR-based figure
you might see in a passage on the biology
and biochemistry section of your MCAT,
While you would normally be provided
with more context and information,
we'll ignore that information for the sake
of simplifying this figure.
So when it comes to interpreting any sort
of graph presented to you on the MCAT,
one of the first things to consider
is breaking down the information presented
on each axis of the figure.
We'll go into the TAID P method in just a few seconds.
But first we want to point out
some PCR-specific information.
So here, the Y-axis shows the mRNA expression
of several different mRNA transcripts relative
to a control, right?
That's why we see it divided by beta-actin.
Beta-actin is a highly
and consistently expressed transcript,
which is why it's a helpful control for this experiment.
In this figure, let's now go ahead
and apply the TAID P method.
First, the title keys us in
that we're looking at our RT-qPCR analysis
of pro inflammatory cytokine expression
in the lungs of hamsters under certain conditions.
Now, let's take a look at our axes.
The X-axis appears quite complicated,
but the researchers have nicely laid out several sets
of independent variables for us,
as denoted by the plus and minus signs.
For example, looking at the tall purple bar
in all three graphs,
we see that they're acid positive, spike-6p positive,
and B38-CAP negative.
The passage related to that figure
would have provided more information
on what exactly the acid spike-6p and B38-CAP mean.
We already discussed the Y-axis and dependent variable,
but again, we're looking at the mRNA expression
of several pro-inflammatory cytokines relative to a control.
The pro-inflammatory cytokines we're looking at here
are IL-6,TNF-α and CXCL 10.
Finally, the most important part of TAID P
are there any patterns that you see?
Fortunately, these graphs have given us bars
that denote statistical significance
which makes our job much easier.
Given this, you can quickly observe
that the purple bar is the tallest
across all three conditions.
And it has some statistical significance
relating to a couple of other conditions.
Beyond that, on test day,
simply keep this information
somewhere in the back of your mind
and keep moving through the passenger onto the questions.
If a question asks specifically about those figures,
you'll be able to quickly pull that information back into
the front of your mind and go back
and quickly analyze the figure.
We've talked a lot about polymerase chain reaction
for the end cap.
Let's now think about how to approach PCR related questions.
When answering questions about PCR,
There are two common pitfalls to keep in mind,
and this is something we emphasize to all of our students.
First, keep in mind that PCR can only tell you information
about the nucleic acid that's being quantified.
For instance, RT-qPCR can only tell you how much mRNA
is being transcribed.
The results of PCR can't necessarily tell you
how much protein is being translated from that mRNA.
This is because the actual ribosomal translation
of the mRNA might be upregulated
or mRNA that's being produced is also being rapidly degraded
as such the amount of mRNA
doesn't give us any additional information
as to the protein expression.
This is a very key point relating back to the central dogma
and the test writers love to trip students up here,
so be careful.
Second, be sure to keep an eye
on the levels of statistical significance,
this will be true for most of the quantitative figures
presented on the end cap.
If there are no symbols
that indicates statistically significant differences
and the quantities that are presented in the figure
may not indicate anything meaningful.
PCR or polymerase chain reaction
in related experimental techniques
often appear in the biology and biochemistry section
of the MCAT.
Throughout this video, we've learned what PCR is
and how to interpret any results and figures.
We've also discussed real time
quantitative polymerase chain reaction
and how you need to understand
that experimental technique for the MCAT.
We hope this video has been a helpful resource for you
and your cat study.
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