Polymerase chain reaction (PCR) expedites recombinant DNA technologies, which involve the insertion of a DNA sequence into a plasmid vector  or the genetic material from another organism. PCR allows the isolation of a defined DNA sequence by amplification of a specific region of target  DNA. PCR-amplified DNA can be directly used for cloning, sequencing, and generating probes for gene expression analysis. PCR enables analysis  of unknown DNA sequences from very small amounts of starting material. PCR allows real-time quantification of the amount of a given DNA  sequence in a complex sample. PCR also provides a useful tool for diagnosis of diseases and detection of infectious agents, such as bacteria and viruses.

PCR Reaction

A typical PCR reaction contains the template DNA, a thermostable DNA polymerase, two small DNA oligonucleotides (or primers), and dNTPs. The reaction usually takes three major steps:

(1) The sample is heated to 94–98°C, which denatures the double-stranded template DNA, separating it into two single strands;

(2) The temperature is usually decreased to 50-70°C, allowing two primers to anneal to the specific sequences of a template DNA at each end;

(3) The temperature is increased to 72°C, allowing the DNA polymerase to extend the primers by the addition of dNTPs to create a new strand of DNA, thereby doubling the quantity of target DNA.  

The cycle of denature, annealing and extension is repeated for 20-40 times, resulting in the exponential replication of a specific target DNA sequence. For example, a 20-cycle PCR reaction yields about 1.05 million of copies of a target DNA, while 30-cycle PCR gives over 1 billion of copies by calculation. A PCR reaction occurs in three phases:

(1) The exponential phase is the period in which exact doubling of DNA product occurs every cycle. Real-time PCR quantitation is carried out during this phase;

(2) The linear phase occurs as the reaction is slowing due to the consumption of the reagents which become limited;

(3) The final stage is the plateau phase, which occurs when no additional amplicon is generated.

Thermostable DNA Polymerases

PCR employs a thermostable DNA polymerase that is heat resistant and capable of generating new strands of DNA using a template DNA and primers.  The first described thermostable DNA polymerase is Taq DNA polymerase isolated from bacterium Thermis aquaticus.  However, the lack in 3’ to 5’ exonuclease proofreading activity of Taq results in a high error rate when replicating DNA by PCR. High fidelity PCR is required for many applications, including cloning and sequencing, where sequence accuracy is crucial. It is recommended that low error rate enzymes, such as Pfu DNA polymerase from Pyrococcus furiosus, should be used in order to reduce spurious mutations introduced during PCR.  Although it possesses a 3’ to 5’ proofreading activity, Pfu exhibits moderately 5 to 10-fold lower error rate than Taq.  To eliminate spurious mutations in particular for long amplicons, a thermostable DNA polymerase with exceptional fidelity is required. 

G&P High Fidelity (HiFi™) DNA Polymerase

We developed G&P HiFi™, an engineered, proof-reading thermostable DNA polymerase that exhibits exceptional fidelity, sensitivity and robustness. Featuring an error rate over 100-fold lower than Taq, it is the most accurate thermostable DNA polymerase available for high fidelity PCR.  The feature makes G&P HiFi™ a superior choice for cloning, sequencing, mutagenesis, and other molecular biology applications requiring high fidelity. G&P HiFi™ DNA polymerase plus the optimized buffer system offers several unique features in comparison with other common high fidelity PCR enzymes:

• Highest Fidelity – Over 100x improvement comparing to Taq DNA polymerase

• Superior Robustness – Amplification of a wide range of amplicons (up to 15 kb)

• Extreme Sensitivity – Higher yields with lower amounts of template & enzyme 

» To learn more about these features, please click here: G&P HiFi™ PCR System 

PCR Optimization

PCR can fail for many reasons, due to its sensitivity to the quality and quantity of template or contamination, causing amplification of spurious DNA products.  A number of strategies are developed for optimizing PCR.  Contamination with extraneous DNA can be addressed with laboratory procedures that separate PCR mixtures from potential contaminants.  This usually involves spatial separation of PCR-setup areas from others and thoroughly cleaning the work surface between reaction setups.  Proper primer-design is also important in improving PCR success rate and in eliminating spurious products.  The usage of PCR additives can help with amplification of complex templates or problematic regions of target DNA (e.g., GC-rich DNA). 

The amplification of GC-rich DNA by PCR is often problematic due to stable secondary structures in the target DNA that are resistant to melting. The complex secondary structures cause DNA polymerases to stall, resulting in incomplete or non-specific amplification. Various methods and additives have been developed to facilitate template denaturation.  G&P HiFi™ DNA polymerase is suited for the amplification of GC-rich DNA.  The unique properties of this enzymes include high fidelity, extreme robustness and improved tolerance to DNA melting agents. G&P HiFi is supplied with proprietary reaction buffers and allow for the efficient amplification of GC-rich DNA using longer extension times of 20 – 30 sec/kb per cycle in the presence of PCR additive such as DMSO (3-5%).

"Hot Start" PCR

Hot start PCR is a technique that reduces non-specific amplification during the initial set up stages of the PCR. It may be performed manually by heating the reaction components to the denaturation temperature (e.g., 95°C) before adding the polymerase. Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that dissociate only after a high-temperature activation step. Hot-start or cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature. In general hot start condition is not required for G&P HiFi™ PCR systems that exhibit negligible enzymatic activity in the presence of supplied reaction buffers at ambient temperature.

» Go to the next topic: PCR-mediated Cloning and Mutagenesis

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