Choosing the right DNA polymerase for your PCR

Success of the polymerase chain reaction (PCR) largely depends on the choice of the appropriate DNA polymerase. DNA polymerase is one of the major components needed for setting up a PCR. For PCR, a thermo-stable DNA polymerase is essential, so that it can endure higher temperatures during the cycling conditions. Therefore, thermo-stable DNA polymerases serve as a key player in the current methods of DNA amplification and sequencing.

Based on their amino acid sequences, DNA polymerases are categorized into six families: A, B, C, D, X and Y. Thermophilic enzymes are found in all the six families. Same reaction is catalyzed by all DNA polymerases, that is, adding nucleotides to the 3′-end of the DNA primer to synthesize the new DNA strand complementary to the template DNA. The thermo-stable DNA polymerases synthesize DNA in a template-directed manner, and require primer-template hybrid to begin the synthesis.

Development of the PCR resulted in advancement of countless molecular biology techniques. Following PCR, a large number of various subsequent experimentation can be done. PCR for different purposes require different types of DNA polymerases. PCR is generally done for screening of the recombinant clone or for just simply determining the size of a particular DNA fragment. This can be carried out with simple DNA polymerases. But for cloning the DNA fragment for its expression, library construction, genome walking, RACE (Rapid Amplification of cDNA Ends) etc., different kinds of DNA polymerases are required.

Therefore, selecting an appropriate DNA polymerase, in accordance to the application, is extremely significant for the success of the experiment. DNA polymerases possess following four basic properties on the basis of which suitable DNA polymerase can be selected:

· Thermosatability

For the amplification procedures, DNA polymerases should be thermo-stable essentially. During each cycle of the PCR reaction, a denaturation step (at approximately 95°C) is there to separate two strands of the DNA molecule for next round of synthesis. Therefore, DNA polymerase should be durable enough to tolerate such a high temperature without losing its activity.

Half-life of a DNA polymerase at a specific temperature determines its thermostability. Its survival in a particular procedure largely depends on the factors like reaction mixture, protein concentrations and other reaction conditions.

· Extension rate

The number of dNTPs added per second per molecule of DNA polymerase is known as the extension rate. It highly depends on the reaction mixture and DNA templates. Sometimes there is formation of structures in the DNA molecules, as a result of which primer elongation ceases.

Generally, higher extension rates of DNA polymerases are desired. This facilitates amplification of longer DNA fragments.

· Processivity

Processivity is the probability that a DNA polymerase will not detach from the DNA after the attachment of a nucleotide, while translocating to the next position. It indicates the average number of the nucleotides that a DNA polymerase adds in a single binding event.

Similar to extension rate, processivity depends on the components of the reaction mixture and the sequence of the DNA template. On heterogeneous templates, processivity of each template position depends on the salt concentration.

· Fidelity

Fidelity denotes the frequency of insertion of the correct nucleotide per incorrect insertion. It actually refers to the ability of the DNA polymerases to insert correct nucleotides. It is an intrinsic property of the DNA polymerases. Therefore, DNA polymerases having low efficiency of correct nucleotide insertion, i.e. inefficient DNA polymerases, exhibit low fidelity, whereas, efficient polymerases exhibit high fidelity.

On the basis of the above mentioned properties, different types of DNA polymerases are available commercially. In accordance to the requirements and experiments, appropriate DNA polymerase can be selected from the following classes:

1) Taq DNA polymerase

Taq DNA polymerase was purified from bacterium Thermus aquaticus, found in hot springs. It is the most commonly used thermophilic DNA polymerase that catalyzes template-directed synthesis of DNA using nucleotide triphosphates.

It is used for general purpose PCRs, such as colony PCR (for screening of the recombinant clones), amplification of the DNA fragment for estimating its size, simple detection of the amplified product, PCR based molecular marker studies, etc.

They generally produce DNA fragments with ‘A’ overhang at 3’-end. This means that whenever a DNA fragment will be amplified with Taq DNA polymerase, the amplified products will have a single adenine base at 3’-terminal. Thus, the amplified DNA fragment can be directly cloned into T/A cloning vector.

Properties

· Taq polymerase possesses maximum catalytic activity at 75-80°C. It has half-life of 1.6 hours at 95°C.

· It has a extension rate of 1kb/minute.

· It has been observed that Taq polymerase dissociates from the DNA after attaching approximately 40 nucleotides,. Hence, exhibiting a good processivity.

· It lacks 3’ to 5’ exonuclease activity. As a result of this, it generally has higher error rate and less fidelity. It has error rate between 1X10^-4 to 2X10^-5 errors per base pair.

Limitations

Since Taq polymerases have higher error rate, it cannot be used for amplification of DNA fragments where high accuracy is desired. For example, it cannot be used for amplifying the DNA fragments to be cloned and expressed, and for mutagenesis studies.

2) Proofreading DNA polymerases

DNA polymerases are said to be proofreading when they possess 3’ to 5’ exonuclease activity. Whenever there in incorporation of non-complementary nucleotide in the growing DNA strand, the proofreading DNA polymerase by the virtue of its 3’ to 5’ exonuclease  activity, removes the erroneously attached bases by hydrolysis. This is an irreversible reaction. It significantly, increases the accuracy of the DNA synthesis from the template DNA, thereby exhibiting high fidelity.

Because of high fidelity, they are extremely useful for techniques that demand high accuracy in the DNA synthesis. For instance, for cloning and expression of amplified product, mutagenesis studies, etc. proofreading enzymes are obvious enzymes to choose.

They usually generate blunt-ended PCR products. Therefore, PCR fragments amplified by proofreading polymerases can be directly used for blunt-end cloning.

Properties

· Exhibits maximum catalytic activity at 68-75°C and have superior thermostability. Their half-life is 6.7 hours at 95°C.

· Relatively slower than Taq polymerase. Extension rate is approximately 0.5kb/minute.

· Their processivity is quite low as compared to Taq polymerase, i.e. approximately 4-30 nucleotides.

· Possess 3’ to 5’ exonuclease proofreading activity. They have far less errors as compared to Taqpolymerase. Their error rate is approximately 1.5 X 10^-6 error per base pair. This provides 5-15 fold higher fidelity than Taq polymerase.

Limitations

They are quite slower and therefore require more time to amplify the DNA fragments. Since they have low processivity, very high optimization is required. For this, generally gradient PCR is done.

Example

· Pfu DNA polymerase (Stratagene).

· Vent_R® DNA polymerase (New England Biolabs).

3) Polymerases for the amplification of long templates

Generally, Taq polymerase is able to amplify DNA fragment of ~3kb only. Whereas, the proofreading DNA polymerases can amplify PCR products of size up to ~6kb. Therefore, in order to amplify DNA fragments of much larger size, several DNA polymerases have been developed. Generally, they are mixture of a Taqpolymerase and a proofreading polymerase.

They are extremely useful for producing high yield of PCR product from genomic DNA with accuracy. They can amplify fragments as large as 30kb in size.

Properties

· Highly thermostable similar to Taq polymerase. Optimum temperature is usually 68°C.

· Extension rate is little bit higher than Taq polymerase, i.e. ~1.5kb/minute.

· Processivity similar to Taq polymerase.

· Due to inherent 3’ to 5’ exonuclease proofreading activity, they are 3-fold more accurate than Taqpolymerase.

Limitations

Since it is a mixture of Taq polymerase and proofreading DNA polymerase, its fidelity is not very high.

Examples

· Expand long template PCR system (Roche)

· LongAmp® Taq DNA polymerase (New England Biolabs).

4) Hot start polymerases

Hot start PCR is a modification of conventional PCR. It is used mainly to suppress non-specific product amplification and to increase the yield of the desired product.

In conventional PCR, the Taq polymerase remains active at room temperature and even on ice, to some extent. At this point, when all reaction components are mixed, primers can anneal non-specifically to the template DNA. The non-specific annealed primers can be extended by Taq polymerase, resulting in accumulation of non-specific products, thereby decreasing the yield of desired product.

Therefore, in hot start PCR, the DNA polymerase is kept inactive with the help of neutralizing monoclonal antibody until higher temperatures are reached. When the temperature raises, the antibody dissociates from the enzyme and gets inactivated making the DNA polymerase active, it considerably reduces non-specific priming, primer-dimer formation and, thus, increases the product yield.

They are very useful when the amount of DNA template is very low (i.e. less than 10⁴ copies of template DNA), when the DNA template exhibits high complexity (e.g. mammalian genomic DNA), and when several pairs of primers are there in the PCR (e.g. multiplex PCR). In all such cases, hot start PCR works best in combination with ‘touchdown PCR’ protocol.

Properties

· Remains inactive until higher temperatures are attained.

· Their extension rate and processivity is similar to that of Taq polymerase, since they are basically Taqpolymerase bound with an antibody.

· Lacks 3’ to 5’ exonuclease proofreading activity. Some suppliers provide blend of proofreading polymerase along with it to provide 3’ to 5’ exonuclease activity.

Limitations

Have lower fidelity and thus higher error rate.

Examples

· Advantage® 2 PCR enzyme system (Clontech).

· AccuStart^TM Taq DNA polymerase (Quanta Biosciences).

2) Next Generation Polymerases

Next generation of polymerases have been developed to overcome the shortcomings of the existing proofreading polymerases. They are engineered and generally are Pfu-based DNA polymerases. They are incorporated with several characteristics that provide them increased processivity, high fidelity and extreme speed.

By the use of high affinity DNA binding domain, their processivity has been increased dramatically. With the help of this domain, DNA polymerase can anchor much better. This prevents its early dissociation from the template DNA. Therefore, there is incorporation of more nucleotides per binding event due to improved processivity. This enhances PCR yield and shortens the extension time.

They are very suitable for high performance cloning in very short time. Their improved speed makes them desirable for higher throughput. They also provide very high yield of the PCR product. They are ideal for difficult targets and provide accurate results even with complex DNA templates having very high GC content (i.e. >80%).  They can amplify DNA fragments up to ~10kb. They are highly sensitive and can use very low amounts of DNA for amplification.

Properties

· Highly thermostable, as developed from Pfu polymerases.

· Modified to generate PCR fragments in much shorter extension times. With some next generation DNA polymerases, extension rate is as high as 1Kb/15-30second.

· Their processivity is 12-fold higher than the Pfu DNA polymerases.

· Highly accurate. Exhibits robust performance and reliability.

Limitations

They are relatively expensive.

Examples

· Herculase® II Fusion DNA polymerase (Stratagene).

· Phusion® DNA polymerase (Finnzymes).

Hence, from above discussion it is clear that for routine and general PCR you should use Taq DNA polymerase, whereas, if you have to express a gene or carry out mutagenesis experiment, you should go for the proofreading or new generation DNA polymerases. Similarly, if you need to carry out RACE or Genome Walking experiments, a Hotstart polymerase should be your pick. In addition, if you want to amplify large template then you should choose Expand Long DNA polymerase.