Successful PCR depends on two crucial components, an optimized reaction buffer, and a high-quality, thermostable DNA polymerase (such as Taq DNA polymerase).
Four basic properties of DNA polymerases can help you define the best enzyme for your particular research needs:
1. Thermal stability. A denaturation step at approximately 95°C in each PCR cycle separates the two strands of a DNA molecule. DNA polymerase must be robust enough to tolerate high-temperature cycles without compromising activity, a factor dependant on buffer composition and pH.
2. Extension rate. This refers to the speed at which nucleotides are added, per second, per molecule of DNA polymerase, a factor determined by extension temperature, DNA template sequence and buffer composition. Early polymerases exhibited extension rates of about 1 kb per minute at 72°C, but contemporary enzymes are generally faster (approximately 4 kb per minute).
3. Fidelity. Fidelity is an inherent DNA polymerase property defining the frequency of insertion of an incorrect nucleotide per kb of DNA. For standard polymerases, fidelity refers to the ability to discriminate correct vs. incorrect nucleotide incorporation and can be influenced by the buffer composition. High-fidelity polymerases are more accurate because of the ability to "proofread" and excise incorrectly incorporated mononucleotides, replacing them with the correct base.
4. Processivity. The probability that a polymerase will detach from DNA during extension, indicating the average number of nucleotides the enzyme adds in a single binding event, is known as its processivity. Like extension rate, processivity depends on buffer composition (salt concentrations) and the sequence of a DNA template. High processivity is important when amplifying long amplicons.
Different configurations of these four variables have produced different classes of DNA polymerase, namely:
Standard thermostable DNA polymerases
These polymerases are suitable for routine PCR, such as detection of amplified product and estimation of product size. Standard Taq produces fragments with a single-based ‘A’ overhang at the 3’-end, enabling direct insertion into T/A cloning vectors. Newer Taq DNA polymerases exhibit good processivity and fast extension rates but lack proofreading capabilities and so cannot be used for amplifying fragments for cloning and expression, or for mutagenesis studies.
Hot-start (HS) polymerases
Hot-start DNA polymerases are used to suppress nonspecific product amplification during setup to increase yield of the desired product. This is because standard Taq can be active even on ice (4°C). When reaction components are mixed, primers can anneal nonspecifically to each other or to the template DNA. Taq can extend those nonspecific annealed primers, resulting in accumulation of nonspecific products and decreasing yields.
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With hot-start PCR, the DNA polymerase is inactivated by a chemical modification or antibody that dissociates and becomes inactive when the temperature is raised. This greatly reduces nonspecific priming and primer-dimer formation and increases product yield. As chemical hot-start can require up to a 10-minute inactivation step, antibody hot-start is used for Fast PCR, which requires less than two minutes for inactivation.
Hot-start is useful when DNA template amounts are low, DNA templates are highly complex or several pairs of primers are used, as in multiplex PCR. Newer hot-start enzymes also exhibit good processivity and fast extension rates, but they lack proofreading ability and so cannot amplify DNA fragments for subsequent cloning or expression, or for mutagenesis studies.
High-fidelity polymerases (Hi-Fi)
Proofreading DNA polymerases possess a 3’-to-5’ exonuclease activity and remove erroneously attached bases when incorporated in the growing DNA strand. This increases the accuracy of DNA synthesis from template DNA, resulting in a better DNA copy. For cloning and expression of amplified product, mutagenesis studies and related applications, proofreading enzymes should be used.
But be advised: Hi-Fi polymerases also have an endonuclease activity that removes the ‘A’ overhang at the 3’-end, so PCR fragments amplified by proofreading polymerases require blunt-end cloning. These polymerases also tend to have lower speed and processivity rates. Processivity can be increased through addition of a high-affinity DNA binding domain that helps the polymerase anchor and prevents early dissociation; however, these polymerases tend to be comparatively expensive.
Polymerases for amplification of long amplicons
Amplification of long amplicons requires combining the processivity of standard DNA polymerases with the accuracy of a proofreading polymerase. This is achieved by blending the thermostable polymerase with a proofreading enzyme. Adding an optimized buffer, usually with high salt concentration, results in high-yield PCR products from genomic DNA and amplicons as long as 25 kb in size.
In conclusion, you should choose your PCR enzyme as follows:
- For general and routine PCR, use an ordinary, standard thermostable DNA polymerase, such as Taq.
- For gene expression or mutagenesis experiments, use a proofreading enzyme.
- For clean product and high yield, use a hot-start polymerase.
- For long amplicons, use a long-range DNA polymerase.
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