Welcome to Applied Biological Materials’ PCR video series! In this video,
we will introduce the basics of PCR. Polymerase chain reaction, or PCR is a technology
indispensible to genetic and molecular biology. Many of its applications are widely used in
the world around us, including DNA sequencing, DNA fingerprinting, forensics, detection of
microorganisms, and diagnosis of hereditary disease. PCR allows fast and inexpensive amplification
of DNA fragments. Because it is able to generate large quantities of DNA from a small amount
of nucleic acid, PCR is also referred to as molecular photocopying.
PCR depends on a series of 20 to 40 repeated cycles of DNA replication by a DNA polymerase
enzyme. After each cycle, the number of DNA strands is doubled, and at the end of a 40
cycle reaction, more than 1 trillion copies are generated from a single copy of the DNA
molecule. The PCR process is separated into four steps:
initialization, denaturation, annealing, and elongation. In the first initialization step,
the reaction is heated to 94–96°C for 2 to 10 minutes to activate the DNA polymerase
and to denature other contaminates in the mixture. In the case of colony PCR screening,
this step also facilitates cell lysis to release DNA and denature other cellular proteins.
In the denaturation step, hydrogen bonds between the double-stranded DNA are broken by heating
the reaction to 94–98°C for 20–30 seconds. After the DNA strands are separated, primers
bind to a complementary sequence in the DNA template to guide DNA polymerase replication.
In this step, the reaction temperature is lowered to 50-65°C for 20–40 seconds to
allow for optimal primer annealing. After the primers establish a starting point for
the enzyme, DNA polymerase starts to incorporate dNTPs in a 5’ to 3’ direction to synthesize
a new DNA strand. The temperature and extension time for this elongation step depend on the
type of DNA polymerase enzyme and the target amplicon. Commonly used Taq Polymerase polymerizes
at a speed of 1-1.5 kilobases per minute and works ideally at 72-78°C. The cycle then
repeats from denaturation to elongation 20 to 40 times. At the end of the last cycle,
there is a final elongation step that keeps the reaction mixture at 72-78°C for 5-15
minutes. This ensures that any remaining single-stranded DNA is fully extended after the last PCR cycle.
A final holding step keeps the PCR at 4-15°C for an indefinite time, keeping the products
for short-term storage. The most common DNA polymerase enzyme used
in PCR is Taq polymerase, a thermostable DNA polymerase that allows multiple cycles of
amplification without the addition of new enzyme after each denaturation step. Since
the initial discovery of Taq DNA polymerase, many variations of the Taq enzyme have been
engineered with different attributes that can be utilized for specific PCR applications.
For example, we at Applied Biological Materials, also known as abm, carry a modified Taq enzyme
that has decreased error rates. Also, abm has engineered a robust Taq enzyme that amplifies
DNA directly from blood samples. There are many more engineered Taq enzymes and other
DNA polymerases available. To visualize the DNA fragments after amplification
by PCR, gel electrophoresis is used. By comparing the gel bands to a molecular weight marker,
researchers can estimate the size of the amplified products to know if their desired genes were
successfully amplified. Many factors can interfere with a PCR reaction.
Some are easy to eliminate while others are trickier to manipulate and would require experience
to tackle. In general, factors that are important in PCR include the nucleotide composition
of the DNA template, DNA polymerase enzyme choice, buffer components, primer design,
additives and inhibitors. Depending on the sequence of the target DNA,
different strategies may be needed for a successful PCR. For example, GC rich templates have more
hydrogen bonds compared to AT rich templates. This results in incomplete strand separation
which may cause the PCR to fail. In addition, high GC content templates tend to lead to
more secondary structures, which can arrest the polymerase leading to pre-mature termination.
In these cases, secondary structure destablizers such as DMSO can be added.
At the same time, AT rich templates also require special attention because of their low primer annealing
temperatures. Non-specific primer annealing can occur under low annealing temperature,
and primer specificity can be retained using a combination of lowered extension temperature
and the use of additives such as TMAC. Long templates are difficult to amplify because
of their higher likelihood of DNA template being broken or degraded by depurination.
Depurination refers to a chemical reaction during which the β-N-glycosidic bond in the
purine nucleoside is cleaved to release a nucletide base due to hydrolysis. As Taq DNA
polymerase will not extend through apurinic positions during replication, depurination
is an important limiting factor of long template amplification. This can be avoided by using
a higher pH reaction buffer and decreasing the denaturation time to limit depurination,
or by using a proofreading DNA polymerase. As mentioned earlier, there are many variations
of DNA polymerases commercially available, each with different features. Depending on
the DNA template, choosing the correct DNA polymerase, and pairing it with an appropriate
buffer can be critical for a successful reaction. Apart from DNA polymerase choice, having a
good pair of primers can also dictate the outcome of a PCR. Some considerations to keep
in mind include the length of the primer, the annealing temperature, and the sequence
of the primer. For example, the pair of primers should not have complementary sequences; otherwise
they can easily lead to self-dimer or primer dimer formation and compete with template-primer
annealing. Many naturally occurring substances, such
as polysaccharides, tannic acid and EDTA, can also inhibit PCR. Some inhibitors may
degrade or modify the DNA template, while others can disturb the annealing of primers
to DNA or alter the DNA polymerase activity. Inhibitors can be removed with sample-specific
nucleic acid isolation protocols, or sometimes the use of additives can help in reducing
the inhibition. For a complete list of PCR additives and their mechanisms of action,
please refer to our knowledge base. A combination of favorable factors contributes
to a successful PCR. Taking all these factors into consideration, abm provides a wide range
of DNA polymerases, and has formulated optimal PCR MasterMixes for each polymerase. We also
offer kits that make DNA amplification from plants, tissue and blood samples easy! Check
out our comprehensive list of PCR products in the link provided below. Thank you for
watching and make sure you follow our next video on the variations of DNA polymerases.
Don’t forget to leave your comments and questions below.