Welcome back to Applied Biological Materials PCR video series. In this video, we will cover
the structure, important characteristics and the different variants of DNA polymerase.
DNA polymerases are enzymes responsible for assembling nucleotides to create new DNA molecules.
These DNA polymerases are indispensable to cell divisions as they duplicate the genetic
information that would be passed to the next generation of daughter cells. One of the most
fundamental technologies in genetic and molecular research is the Polymerase Chain Reaction
also known as PCR. PCR utilizes the ability of DNA polymerase to replicate DNA strands by amplifying a large
amount of DNA from a small amount of nucleic acids. To learn more about the principles
of PCR, we invite you to watch our PCR introduction video before continuing with this one.
Throughout the years, scientists have utilized different variants of DNA polymerases for
different applications. All these DNA polymerases have a similar shape, in that they resemble
a “right hand” with common structural features such as a “thumb,” “palm,”
and “fingers”. The “palm” is associated with catalysis of the phosphoryl transfer
reaction. The “fingers” are involved in the interactions between the nucleoside triphosphate
being inserted and the existing template base. And lastly, the “thumb” is to assist in aligning
the double-stranded DNA. Primers are oligonucleotides that can bind
to a specific sequence of the DNA template to guide DNA polymerase replication. As the
DNA stand separates, a primer with a free 3’ hydroxyl group anneals to its specific
template sequence, triggering the DNA polymerase to add free deoxynucleotides onto the hydroxyl
group, via phosphoryl transfer reaction to elongate the new strand in a 5’-3’ direction.
When a deoxynucleoside triphosphate (dNTP) binds to DNA polymerase, the enzyme undergoes a
conformational change so that only the base on the template strand can have a complementary
nucleotide fit, such as cytosine to guanine, and thymine to adenine.
There are two characteristics that define a polymerase,
fidelity and processivity. Fidelity refers to the accuracy of the DNA polymerase during
replication of the amplicon; high fidelity is achieved by having a proofreading mechanism and a low mis-incorporation rate.
There are 3 types of domains a polymerase can have.
First is a 3’-5’ exonuclease domain which allows them to remove mis-incorporated nucleotides
from the 3’ end while the DNA is being formed in the 5’-3’ direction. Second is 5’-3’
exonuclease domain, that aids in primer removal and nick repair.
Some DNA polymerases lack any exonuclease activity, which allows the extension of a DNA strand
at the 3’ end to displace the downstream DNA. Fidelity can be affected by the PCR buffer
components and thermocycling conditions. For example, having unequal nucleotide
concentration, and heating the DNA template to high temperatures will lower
the fidelity of DNA polymerase. The second characteristic is Processivity.
Processivity is the number of nucleotides a polymerase is able to incorporate before
dissociating. Majority of DNA polymerases are low in processivity as they bind to the
template and dissociate, however, DNA polymerases with high processivity are able to extend
as far possible, adding several nucleotides per second. DNA clamps are multimeric
proteins that interact with DNA polymerase, increasing processivity. DNA clamps slide
along the DNA strand and interacts with the polymerase to stimulate association between
the polymerase and the DNA strand facilitating a timely replication. Scientists have increased
processivity in DNA polymerase by fusing it with a DNA binding domain, maximizing PCR
yield and speed. Similar to fidelity, processivity is affected by buffer conditions.
Working along with processivity is the extension rate. The extension rate refers
to the speed at which the nucleotides are added per molecule of DNA polymerase during
extension. It is also proportional to the processivity of the DNA polymerase. Extension temperature,
buffer conditions and template sequences can affect the extension rate. As well, thermal stability
is essential for the DNA polymerases to maintain their stabilities at high temperature in PCR.
Thermal stability can also be measured by the half-life of DNA polymerases which affect its ability to retain its activity under sustained
temperature as high as 95 degree Celsius. There are many types of DNA polymerases; therefore
it is important to know the characteristics of each type in order to choose the most suitable
enzyme for your experiment. As well, all DNA polymerases are derived from organisms that
have optimal growing temperatures from mesophilic to ultra thermophilic.
There is a variety of polymerases that are more suitable depending on what you want to achieve,
such as the Bst DNA Polymerase, which is suited for loop-mediated isothermal amplification and the Bsu DNA Polymerase
which is suited for isothermal strand displacement applications. Taq DNA polymerase is the most
commonly used enzyme with the 5’-3’ exonuclease activity. The 5’-3’ exonuclease activity
produces DNA with sticky ends and the PCR products can be used for TA cloning. Polymerases
that elicit 3’-5’ exonuclease activity is correlated to a higher fidelity, such as
Pfu, KOD, Long-Range, and Pwo DNA Polymerase. With a proofreading mechanism and a low mis-incorporation rate,
polymerases with high fidelity will generate blunt ended products.
If you wish to increase product yield by reducing nonspecific amplification, it is best to use Hot-Start
DNA Polymerase. In Hot-Start PCR, the DNA polymerase, usually Taq or pfu polymerase
is chemically modified or antibody bound to remain inactive during lower annealing temperatures.
When heated, during the denaturation step, the chemical or antibody inhibitors become
inactive or they dissociate from the DNA polymerase. This activates the DNA polymerase to start incorporating nucleotides.
At abm, we offer a wide selection of PCR products, including the polymerases
that are mentioned in this video. If you’re interested, a link is provided below showcasing
our DNA polymerases. For more information on PCR please check
out our knowledge base. And did you enjoy watching
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