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Welcome back to BOGObiology! In this video we'll
be discussing Polymerase Chain Reaction or "PCR".
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We'll be learning about what PCR is, how it's used,
its reagents the steps in the PCR process, and how
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PCR is used in covid testing. PCR or "polymerase
chain reaction" is a genetic copying process
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used in biotechnology. Biotechnology is a rapidly
growing field that harnesses naturally occurring
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processes for useful purposes. In the case of PCR,
it harnesses the process of DNA replication to
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make copies of genetic material. PCR is sometimes
referred to by the nickname "molecular photocopying".
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PCR makes millions to billions of copies
from a small amount of genetic material.
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The number of copies doubles with each cycle of
the reaction, resulting in exponential growth.
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PCR is an easy way to replicate a small sample so
there is enough to study, analyze, or use in other
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reactions. PCR is used in many industries but the
most familiar are likely forensics, agriculture and
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medicine. In forensics, PCR can be used to amplify
genetic material from an unknown source so that
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it can be compared to a particular suspect
or to a large DNA database. DNA evidence is
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now routinely used to solve crimes. In agriculture,
PCR is a key part of plant genotyping for breeding
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so that desirable traits can be combined, or to
determine if a particular plant should be cloned.
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In medicine, PCR is a diagnostic tool used in
genetic testing, tracking cancer mutations, and
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of course in covid testing. If you're aiming to
perform PCR, several reagents must be combined:
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A DNA sample, a polymerase enzyme,
deoxynucleosides, primers, buffers and cofactors.
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The DNA sample contains the sequence of interest
to be copied. This will become known as the
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template strand. The enzyme DNA polymerase
will be used to build the new DNA strands.
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Most types of DNA polymerase would be denatured
by the hot temperatures used in the PCR reactions.
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So for these purposes, we use a special polymerase
enzyme called Taq Polymerase. This enzyme has been
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isolated from the heat resistant bacteria thermus
aquaticus, and can withstand the hot temperatures
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needed for PCR. Deoxynucleoside triphosphatases or
DNTPs are the building blocks used to construct
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the new DNA copies. Sometimes these are also called
free nucleotides. The Taq polymerase will arrange
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the DNTPs using the template strand as a guide.
Primers are single stranded chunks of DNA that are
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complementary to the start of a target region. They
tell the Taq polymerase exactly where to start copying.
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Because it's so important for the primers to
mark only the correct part of
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the template strand, a tremendous amount of
effort goes into designing them. We need
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to add two additional reagents to give them
the best possible chance of working properly.
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The PCR process also needs two more reagents to
create the ideal conditions for amplification:
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a buffer solution and a magnesium cofactor. Buffer
solutions help to maintain optimal conditions for
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a reaction to occur. Often this means maintaining
the pH, or concentrations of particular ions.
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There are many types of buffers and they're common in biotech.
The magnesium cofactor has two roles; it helps the
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primers to adhere at the correct site and it helps
the Taq polymerase enzyme to function optimally.
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Usually these magnesium ions are added to the
mixture in the form of MgCl2. PCR mimics many
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aspects of the natural process of DNA replication.
DNA is a double-stranded molecule and it needs to
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be replicated faithfully before the cells can
divide. In nature, one enzyme separates the two
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strands and then another creates a complement
to each strand resulting in two identical copies.
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PCR is a very similar process but it uses a machine
called a thermocycler rather than a cell. The steps
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of PCR are denaturation, annealing, and extension,
and the machine uses heat to control the reaction.
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During the denaturation phase, we heat the
DNA to approximately 95 degrees celsius
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(which is almost boiling if you're someone who is used to
fahrenheit!) This breaks the hydrogen bonds in the
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middle of the molecule and creates two template
strands for copying. The second phase of PCR
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is annealing which takes place at about 55 degrees
celsius; much cooler than the denaturation phase.
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During the annealing phase, primers
flank the sequence of interest on
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the template molecule in preparation for
copying. Primers are single-stranded DNA
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fragments that are designed to stick
to a very specific part of the sample.
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It's very important that the primers stick to the
correct spot of the DNA sample because that's the
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area that's going to be copied. Primers are usually
18 to 22 bases in length with a maximum of about
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30. However, finding the correct length is a
balancing act; shorter primers easily stick to
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the template strand but they are also much
more prone to annealing in the wrong place.
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A primer's tendency to stick only in
the correct place is called specificity.
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The annealing stage is where the magnesium
first comes into play. The template strand has
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a negatively charged backbone and the primers
have a negatively charged backbone. These would
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usually repel each other, preventing the primer
from sticking. The positively charged magnesium
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ions keep the primer in the correct orientation
so it can more easily bind to the correct site.
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The buffer also helps with the primer annealing
process; if the primer sticks correctly the buffer
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will stabilize the hydrogen bonds that
form between the template and the primer.
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If the primer happens to adhere in the wrong
place, the buffer will destabilize the bond.
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The third stage is extension which
occurs at about 72 degrees celsius.
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This is where the "polymerase" of polymerase
chain reaction finally comes onto the scene.
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The Taq polymerase enzyme moves along the template
strand from the primer starting point in the five
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prime to three prime direction. Remember that
polymerase can move only in this direction. The
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Taq polymerase creates a new complementary strand
out of the DNTPs that we added to the reaction.
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At the end of the extension phase,
the amount of DNA has doubled.
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Magnesium also plays a role in the extension phase.
It helps to form a bond between the three prime
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end of the primer and the phosphate
group of the first DNTP.
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PCR is a cycle; the denaturation, annealing,
and extension phases repeat over and over,
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doubling the number of copies each time.
After one cycle, there are 2 copies, then 4,
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then 8, then 16, etc. After 25 cycles there will
be 2 to the 25 or roughly 33 million copies.
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Even though the original sample may have been
quite small, if the process has been performed
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correctly there will now be plenty of material for
analysis. PCR has long been used in diagnostics but
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with the rise of the Covid 19 global pandemic,
PCR testing has become far more widely known.
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PCR testing for the virus uses a modified version
of PCR called RTq-PCR also known as "reverse
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transcription quantitative pcr". The name RT-qPCR
sounds like a confusing jumble of random letters, so we're
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going to break it down step by step beginning with
the "RT" portion. Many viruses including SARS-CoV-2
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the virus that causes covid 19, use single stranded
RNA rather than DNA as their genetic material.
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To do PCR on it, we need to add some
additional reagents to the PCR mixture.
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Reverse transcriptase is a naturally occurring
enzyme that can be used to manufacture DNA by
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using RNA as the template. This is the reverse of the
usual DNA to RNA process. We call this copy cDNA,
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short for "complementary DNA". The primers are made
specifically to only adhere to the viral RNA; only
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the viral genetic material if it's present will be
duplicated. Once the cDNA is created from the RNA
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it can be copied using PCR just like regular DNA.
We can't tell whether the viral genetic material
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is present in the tube just by looking at it,
so this is where the "q" or "quantitative" part of
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RT-qPCR becomes important. Like standard PCR, RT-qPCR
makes copies of a specific region of interest
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but it also measures the amount of genetic
material in a sample by using fluorescence.
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Adding a glowing reagent into the DNA allows a
machine to measure or quantify how much DNA is in
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a sample. As the amount of genetic material grows
exponentially, a computer measures and records the
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level of fluorescence on a chart. The amount of
material doubles with each cycle; if the patient's
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sample contained viral RNA, the tube will grow
brighter and brighter as the process continues.
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If the sample contained no viral RNA, the
tube will remain dark. There are two sets
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of fluorescent reagents that are commonly used (as
of the making of this video) SYBR Green and Taqman
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probe. Both reagents have been shown to be equally
accurate in detecting the presence of SARS CoV-2.
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SYBR Green is a dye that attaches to
double-stranded DNA but will not attach to
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single-stranded DNA. As the amount of DNA doubles,
the amount of fluorescence will increase as well.
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In a Taqman probe assay, short sequences called probes
are added to the reaction mixture. They're built to
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temporarily attach to a specific target sequence,
in this case a particular part of the viral DNA.
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The probe contains a glowing reporter
component along with something called
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a quencher that keeps the reporter turned off
as long as the two molecules are close together.
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As the Taq polymerase builds the new
complementary strand, it will dislodge the probe
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and break it into pieces. With the reporter and the
quencher separated, the reporter will start to glow.
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As the number of broken probes grows exponentially
as the reactions progress, the sample will grow
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brighter and brighter. If the level of fluorescence
surpasses a certain threshold, the patient is
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considered to have tested positive. Because the
sample will only start to glow if viral genetic
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material is present, false positives are very rare.
Without viral RNA in the sample, the primers will
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not attach, the reaction will not proceed, and you
won't get any glowing. False negatives, however, do
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sometimes occur. For instance if a patient
is tested quite early after being infected
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there may not be enough viral genetic material
in the patient's sample for it to be detectable.
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That wraps up our discussion of PCR!
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