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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.

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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.

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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.

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PCR-mediated Cloning and Mutagenesis

The isolation and cloning of a defined DNA sequence is a routine task in any Molecular Biology laboratory. There are a wide variety of methods for molecular cloning. One of critical steps is to create cohesive or sticky ends (complementary single stranded overhangs) in both DNA insert and cloning vector, allowing the joint of them in a desired directional manner. Depending on whether specific sequences or sites required for creating such sticky ends, cloning methods can be divided into two major categories, sequence-dependent and sequence-independent cloning. DNA can be assembled together with or without the use of a DNA ligase. Cloning methods can also be classified as ligase-based and ligase-free, or ligation-independent cloning (LIC).

Sequence-dependent Cloning

Sequence-dependent cloning often relies on restriction digestion and ligation, or site-specific recombination. A common strategy involves PCR amplification of a DNA fragment of interest, followed by insertion into a suitable cloning vector. The latter can be achieved in different ways, the most common of which exploits unique restriction sites present in both vector and insert to generate sticky ends, and a ligase to covalently link them prior to transformation into a bacterial host for propagation. This can also be achieved by several LIC methods that utilize an enzyme, such as recombinase and topoisomerase, or enzymes cocktails to yield correct recombinant DNA constructs using site-specific recombination. All these methods are limited by the choice of unique sequences or sites, and by the use of specialized reagents or kits.

Sequence-independent Cloning

Sequence-independent cloning is based on homologous recombination between vector and insert, but does not require specific sequences. It is often referred to as “seamless” cloning because it can avoid introducing additional (or artificial) sequences at the insertion-boundary after cloning. There are increasing demands, largely from Synthetic Biology and Genetic Engineering, for more reliable, efficient and convenient technologies for sequence-independent and ligation-independent cloning (SLIC). High fidelity PCR allows not only for the amplification of a specific DNA sequence, but for the assembly or circularization of multiple DNA fragments containing overlapping sequences at ends. Therefore sequence-independent cloning can be achieved solely by PCR using a single, high fidelity DNA polymerase. G&P HiFi™ is ideally suited for this application, providing an all-in-one solution for sequence-independent cloning.

PCR-mediated Cloning

PCR enables numerous approaches to gene cloning, including TA cloning, LIC, recombinase-dependent cloning, and PCR-mediated cloning. The use of any PCR cloning method is critically based on its reliability, efficiency and simplicity under optimal conditions. Furthermore, the methods should be easy to monitor and optimize. For example, TA cloning and LIC require end modifications that cannot be easily monitored.

Among all the PCR-mediated cloning methods, overlap extension PCR cloning is a simple and reliable way to create recombinant constructs. It can be accomplished essentially by two rounds of high fidelity PCR:

The 1st round PCR to amplify insert or both insert and vector using appropriate forward and reverse primers containing sequences overlapping on one or both ends, as starting materials for next round PCR (see the graphs below);
The 2nd round PCR (overlap extension PCR) to extend overlapping regions between insert and vector to form either a linear, vector:insert fusion (when overlapping sequences present only on one end), a complete circular DNA product (when overlapping sequences present only both ends with a nick on each stand when using a plasmid vector as template), or multimers consisting of repeated vector:insert fusions (when using a linearized vector as template).

The single, linear vector-insert fusion can be circularized using a ligase prior to transformation. In contrast, the circular or multimeric PCR product can be directly used for transformation as the bacterial host will repair nicks or re-circularize the multimer of repeated vector:insert fusions (see the graphs below).

Several variants of overlap extension PCR-based cloning have been described. In general they all use the PCR amplified insert with overlapping sequences to the vector as a mega-primer in the next round high fidelity PCR. However, they differ in template types and conditions for overlap extension PCR.

Circular Polymerase Extension Cloning (CPEC) uses a circular vector as template, which is destroyed by in restriction digestion with DpnI prior to transformation to reduce cloning background.
Prolonged Overlap Extension PCR cloning (POE-PC) uses a linearized vector (generated by resctriction digestion or PCR) as a template for producing DNA multimer using a thermocycling condition with prolonged extension times than standard.

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Both methods allow one or multiple (up to 3) inserts with overlapping regions to be assembled into a vector at any location. However they requires relatively long overlapping sequences (30-50 bp). G&P HiFi™ DNA polymerase is ideally suited for overlap extension PCR cloning due to its superior fidelity and processivity as well as the factor that it does not possess strand displacement activity when using the vector as template. It requires much shorter overlapping sequences (usually 15-20 bp only). Overall, the overlap PCR extension cloning method is easy to monitor and optimize and it does not require restriction endonucleases or DNA ligase. For these reasons, it can be used as a simple and efficient means to create recombinant constructs for the routine study of gene function.

PCR-mediated Mutagenesis

Site-directed mutagenesis is frequently used in protein engineering and structure-function relationship studies. The most common method employs two complementary long oligonucleotides to introduce point mutations (additions, deletions or substitutions) at specific sites of a DNA sequence that has been cloned into a vector. After annealing of both oligonucleotides, which contain mismatched nucleotides at the mutation site, the entire vector is amplified with a DNA polymerase. It produces a nicked, circular DNA, which can then be transformed into a suitable bacterial host for nick repair and multiplication. Site-directed mutagenesis requires the use of a high fidelity DNA polymerase. G&P HiFi™ DNA polymerase is ideally suitable for this due to its high fidelity and robust performance in PCR long amplicons (up to 15 kb). G&P HiFi™ produces high yields of the mutant plasmid with minimal amounts of enzyme, and eliminates spurious mutations introduced during PCR.

References:

1. Appl. Environ. Microbiol doi:10.1128 (2012)

2. Curr Issues Mol Biol 12: 11-16, 2009

3. Biotechniques 48: 463-465, 2010

4. BMC Biotechnology 11: 92-96, 2011

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