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Polymerase Chain Reaction – Variations of DNA polymerase

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Video Summary

DNA polymerases are enzymes responsible for assembling nucleotides to create new DNA molecules. During DNA replication, the polymerase reads the existing DNA strands and semi-conservatively creates new complementary DNA strands. DNA polymerases are indispensable in cell division as they duplicate the genetic information that would be passed to the next generation of daughter cells.

The Polymerase Chain Reaction (PCR), one of the most common and fundamental technologies in genetic and molecular research, utilizes the ability of DNA polymerases to replicate DNA strands in order to amplify large amounts of DNA from a small amount of nucleic acids. For more background knowledge on the PCR technique, please visit our Polymerase Chain Reaction - an Introduction Knowledge Base.



Structure and Mechanism of Action

All DNA polymerases are similar in shape in that they resemble a “right hand” with common structural features such as a “thumb,” “palm,” and “fingers”. The “palm” area is the most similar among the polymerase families, and is associated with catalysis of the phosphoryl transfer reaction (1). The “fingers” domain is involved in the interactions between the nucleoside triphosphate being inserted and the existing template base, and the “thumb” is suggested to assist in aligning the double-stranded DNA (1).

Primers are oligonucleotides that can bind to specific sequences of a DNA template to guide DNA polymerase replication. When the DNA template strands dissociate, a primer with a free 3’ hydroxyl group anneals to its specific template sequence. Researchers can selectively replicate any regions of interest on the template DNA by flanking the region with specifically designed primers. Primer annealing initiates the DNA polymerase to add free nucleotides onto the hydroxyl group via a phosphoryl transfer reaction to elongate the new strand in a 5’-3’ direction. When a nucleoside triphosphate (NTP) binds to DNA polymerase, the DNA polymerase undergoes a conformational change and generates a specific shape/pocket into which only the base of the template strand and a properly shaped complementary nucleotide can fit (cytosine to guanine, thymine to adenine). In this way, DNA polymerase is able to select the correct nucleotides for incorporation (1) (25).


Mechanism of DNA extension by DNA polymerase.


Figure 1 – Mechanism of DNA extension by DNA polymerase.


Fidelity

A DNA polymerase’s fidelity refers to its accuracy during replication of the amplicon; high fidelity is achieved by having a low mis-incorporation rate as well as a proofreading mechanism. Many polymerases have a 3’-5’ exonuclease domain independent of the polymerization process, allowing them to remove mis-incorporated nucleotides from the 3' end while the DNA is being formed in the 5'-3' direction (1). In general, nonproofreading DNA polymerases are sufficient for routine PCR to achieve higher yields, but polymerases with proofreading properties are estimated to have 10-1000X higher fidelity than those without (2). This has major implications for the downstream applications of PCR products for which high accuracy is crucial, such as in mutagenesis and subcloning. Furthermore, polymerases with 3’-5’ exonuclease activity result in DNA products with blunt ends as excess 3’ nucleotides are excised at the end of the extension process (3). This allows for direct ligation into blunt-ended cloning vectors.

All of our DNA Polymerases possess 3’-5’ exonuclease activity.

Mismatched bases


Figure 2 – Mismatched bases are more likely to leave the polymerase site and migrate to the exonuclease site, where the last nucleotide added is removed by hydrolysis.


DNA polymerases can also display 5'-3' exonuclease activity, allowing for the excision of nucleotides from the 5' end of the DNA strand in a nick translation reaction (4). In real-time PCR applications, this attribute is important as it cleaves labelled oligonucleotide probes from the 5' end of the DNA to generate a detectable signal (5).

Conversely some DNA polymerases lack any exonuclease activity, which allows solely for the extension of a DNA strand at the 3' end. This causes displacement of downstream DNA, a property that is exploited in isothermal strand displacement amplification applications (7). (Figure 3)


Mismatched bases


Figure 3 – Mechanism of DNA extension at the 3' end by DNA polymerases lacking exonuclease activity.

PCR originally utilized the Klenow fragment, a proteolytic product of DNA polymerase I isolated from E. coli (6). While it has high fidelity, having retained both polymerase activity and 3’-5’ exonuclease activity, it is irreversibly denatured from the high temperatures used to separate the new strands of DNA (4). New enzyme must be manually added after every cycle - for the typical 30-40 cycles in PCR, this poses a contamination risk and is labour intensive and time consuming. The Klenow fragment has since been replaced by thermostable enzymes from a thermophilic bacterial species, which does not require addition of new polymerase between cycles (4).

PCR buffer components and thermal cycling conditions can affect DNA polymerase fidelity in different ways or to different degrees. For example, while magnesium ions are required as co-factors because they help guide DNA polymerase selection for the correct nucleotide, excess magnesium will reduce the fidelity of DNA polymerases. Unequal nucleotide concentrations will also decrease fidelity as mis-incorporation of the nucleotides available at higher concentrations will occur more frequently. Thermal cycling conditions such as heating of the DNA template to high temperatures for extended duration can lead to the release of bases from the phosphodiester backbone, leading to lowered fidelity.

Processivity

In addition to fidelity, DNA polymerases can be evaluated by their processivity, which is the number of nucleotides a polymerase is able to incorporate before dissociating (9). A polymerase with high processivity binds to the template and extends far and possibly to the end of the DNA template, adding several nucleotides per second. However, most DNA polymerases are intrinsically low in processivity, frequently binding and dissociating from the template and adding one nucleotide per second to produce short DNA product per association/dissociation event (9). In nature, a processivity factor called the DNA clamp is commonly found in organisms to assist the DNA polymerase in the timely replication of a large genome. DNA clamps are multimeric proteins in the shape of a ring that slides along the DNA strand. Its protein-protein interaction withthe DNA polymerase help the association between the polymerase and the DNA strand. DNA polymerases with DNA clamps have dramatically higher processivity and are capable of polymerizing thousands of nucleotides without dissociating from the DNA template. Scientist have also developed DNA polymerases with high processivity by fusing a DNA binding domain to the polymerase, thus maximizing PCR yield and speed, especially for long templates amplifications. (31) (32) Processivity is also affected by the buffer conditions (such as salt concentration) and the sequence of the DNA template(33).

Extension rate refers to the speed at which the nucleotides are added per molecule of DNA polymerase during extension and is proportional to the processivity of the DNA polymerase. Extension temperature, buffer conditions and template sequences can affect the extension rate. A higher extension rate generally leads to reduced thermal cycling time.

Finally, thermal stability is also essential for DNA polymerases to maintain its stability at high temperature during PCR. It can be measured by the half-life of DNA polymerases in the retentio of its activity under sustained temperatures as high as 95°C. It is intimately related to the fidelity and processivity attributes of the DNA polymerase. (34)

Our BlasTaq™ DNA polymerase has an outstanding extension speed of 20-30 sec/kb, making it suitable for quick PCR experiments.

Common DNA Polymerases

Taq DNA Polymerase

Taq DNA polymerase (commonly abbreviated to “Taq pol” or “Taq”) is a thermostable DNA polymerase used in PCR that was originally isolated from Thermus aquaticus, a thermophilic bacteria found in hot springs and hydrothermal vents (10). PCR takes advantage of Taq’s ability to withstand high temperatures required during the denaturation step for strand separation (8). With an optimum activity temperature of 75-80°C, Taq polymerase can be reused through several cycles of PCR without being denatured by the heat itself (8). Taq polymerase also displays high processitivity and can replicate 1 kb of DNA within 30-60 seconds during PCR (11). Taq DNA polymerases also display 5'-3' exonuclease activity, allowing for the excision of nucleotides from the 5' end of the DNA strand in a nick translation reaction (4). This activity is important as it cleaves labelled oligonucleotide probes from the 5' end of the DNA to generate a detectable signal in real-time PCR application. (5)

One of the major drawbacks to Taq is its inability to proofread as it lacks 3'-5' exonuclease activity, therefore giving low replication fidelity (1 error in 9000 base pairs) (11). For routine PCR with short amplicons, or applications in which incorporation of non-standard nucelotides (such as deoxyuridine and inosine) are necessary, Taq’s high processivity and low fidelity is more advantageous. The lack of 3’-5’ proofreading activity also results in a single adenine overhang at the 3' ends of both strands, producing DNA with sticky ends. (3). A potential use for these products is in TA cloning, where the they can be directly ligated into a plasmid vector containing thymine 3' overhangs (12).

TA Cloning


Figure 4 – Mechanism of TA Cloning.

Pfu DNA Polymerase

Pfu DNA polymerase is a thermostable enzyme originally isolated from Pyrococcus furiosus, a hyperthermophilic species of archaea (13). Similar to Taq, Pfu DNA polymerase can be reused throughout several PCR cycles as it operates optimally at 90°C and is not denatured by the heating steps (15). In addition, Pfu DNA polymerase displays 3’-5’ exonuclease activity, and therefore has the ability to proofread by excising mis-incorporated nucleotides, giving it very high replication fidelity (1 error in 1.3 million base pairs) (15) (16).

One caveat to Pfu DNA polymerase’s superior fidelity is its slower speed, as it requires up to 2 minutes to amplify 1 kb of DNA during a PCR cycle (16). Pfu DNA polymerase also produces DNA products with blunt ends, requiring the use of blunt-ended vectors for cloning applications (3).

For applications that require high fidelity DNA polymerase, try our MegaFi™ Fidelity DNA Polymerase which has 13000X higher fidelity than regular Taq.

KOD DNA Polymerase

KOD DNA polymerase is a recombinant form of DNA polymerase derived from the thermophilic solfatara bacterium Thermococcus kodakaraensis KOD1 type strain. KOD DNA polymerase functions optimally at 85°C and displays 3'-5' exonuclease proofreading activity, producing blunt-ended DNA products (21)(22). While KOD DNA polymerase displays high fidelity and processivity for small amplicons, long-distance amplification of amplicons over 5 kb tends to lower product yield due to its strong 3’-5’ exonuclease activity (23). This can be avoided by mixing wildtype KOD polymerase with mutant forms with lower 3’-5’ exonuclease activity, allowing for accurate amplification of amplicons up to 15 kb (23).

Long-Range DNA polymerase

While conventional PCR can be used on amplicons up to 3-4 kb, Taq DNA polymerase is best optimized for amplicons smaller than 2 kb. Taq DNA polymerase lacks 3’-5’ exonuclease activity. It is therefore unable to remove misincorporated bases as it stalls and dissociates without completing the entire sequence (27). On larger amplicons, accumulation of enough mismatches can inhibit PCR, leading to truncated products.

Long-range DNA polymerase is optimized for DNA segments of up to 20 kb. These polymerases combine a thermostable DNA polymerase, usually Taq polymerase (for its high processivity) with a proofreading enzyme containing 3'-5' exonuclease activity to increase fidelity (28). The proofreading polymerase is often derived from a recombinant source and works to remove Taq polymerase’s 3’ mismatches during primer extension (28).

Hot-start DNA Polymerase

Hot-start DNA polymerases are used to increase product yield by reducing nonspecific amplification during PCR setup. Since most DNA polymerases can be active even at room temperature, the combination of reaction components during PCR setup can lead to nonspecific primers annealing to each other or to the template. These nonspecific annealing primers compete for Taq polymerase binding and extension to create undesirable PCR products (29).

In hot-start PCR, the DNA polymerase, usually Taq or Pfu DNA polymerase, is chemically modified or antibody bound to remain inactive during the lower annealing temperature. When heated in the initial denaturation step, the chemical or antibody inhibitors become inactive or dissociate from the DNA polymerase, therefore making the DNA polymerase become active again and free to start incorporating nucleotides.

Hot-start PCR is advantageous for amplifying low amounts of DNA template, highly complex DNA templates, or in multiplex PCR where multiple pairs of primers are used. It can significantly improve the specificity and yield of the product (30), however, because it lacks proofreading activity, the fidelity of hot-start polymerases is limited and makes it unsuitable for applications such as subsequent cloning or mutagenesis.

Our BlasTaq™ HotStart DNA Polymerase eliminates primer-dimer/non-specific amplication and improves yield of the desired product.

Bst DNA Polymerase

Bacillus stearothermophilus is a thermophilic species of bacteria common to soil, hot springs, and ocean sediment environments (17). Bst DNA polymerase is isolated from this species. In addition to its polymerase activity, it displays helicase-like activity to unwind DNA strands (18). Bst polymerase functions optimally at 60-65°C, but denatures above 70°C, making it more suitable for loop-mediated isothermal amplification (LAMP) which does not undergo the high-temperature denaturation steps and thermocycling used in routine PCR (18)(19). While Bst polymerase has 5'-3' exonuclease activity, it cannot proofread as it lacks 3'-5' exonuclease activity (20).

Bsu DNA Polymerase

Bsu DNA polymerase is isolated from the mesophilic soil bacterium <Bacillus subtilis. Bsu DNA polymerase operates optimally at 25-30° and denatures at 75°C, making it unsuitable for PCR. Bsu polymerase cannot proofread as it lacks 3’-5’ exonuclease activity. On the other hand, the large fragment of Bsu polymerase cleaved of its 5’-3’ exonuclease domain can be used in isothermal strand displacement applications.

Tth polymerase

The Tth polymerase is derived from Thermus thermophilus, a thermophilic thermal vent bacterium (25). Tth DNA polymerase functions optimally at 75°C with high processivity, but lacks proofreading 3'-5' exonuclease activity (26). Tth DNA polymerase displays efficient intrinsic reverse transcriptase (RT) activity in the presence of manganese (Mn) ions, allowing it to assemble cDNA from RNA. Because of this property, Tth polymerase can be used for RT-PCR, followed by subsequent amplification of the cDNA product in the presence of magnesium (Mg) ions (26). Tth polymerase produces sticky-ended DNA products (26).

Pwo DNA polymerase

Pwo DNA polymerase is derived from the ultra-thermophilic archaeon Pyrococcus woesei found in deep marine environments (24). Pwo polymerase functions optimally at 100-103°C, and displays high proofreading 3'-5' exonuclease activity, giving it 18-fold higher fidelity than Taq polymerase (15). Pwo polymerase creates blunt-ended products (15)

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