What is PCR??
What are DNA Polymerases??
These are the enzymes used in the PCR.
In this article, I will cover the structure, important characteristics and the different variants of DNA polymerases.
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 nucleicacids.
Throughout the years, scientists have utilized different variants of DNA polymerases for different applications.
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 free3’ 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.
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 canaffect 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’ exonucleaseactivity. 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 activityis 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 fidelitywill 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 DNApolymerase, 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.
That's all about PCR and variants of DNA polymerases.
Stands for "polymerase chain reaction"
What are DNA Polymerases??
These are the enzymes used in the PCR.
In this article, I will cover the structure, important characteristics and the different variants of DNA polymerases.
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 nucleicacids.
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 free3’ 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 canaffect 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’ exonucleaseactivity. 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 activityis 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 fidelitywill 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 DNApolymerase, 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.
That's all about PCR and variants of DNA polymerases.
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