PCR reaction components include purified nucleic acid template, deoxyribonucleotides (dNTPs), oligonucleotide primers and DNA polymerase. Nucleic acid purification and quality assessment are important steps in the PCR experimental workflow. It is important to use proper techniques to isolate and purify nucleic acids from starting material because this step can affect amplification reactions.
DNA polymerases catalyze the addition of dNTPs to an elongating DNA strand by adding bases complementary to the template strand. The identification of thermostable DNA polymerases and subsequent modifications of these enzymes have played critical roles in the advancement and widespread adoption of PCR. The following section provides detailed information about DNA polymerases commonly used in PCR.
Original in vitro PCR amplification of DNA used DNA polymerase isolated from Escherichia coli (Mullis and Faloona 1987). However, this enzyme is irreversibly inactivated when exposed to the high temperatures used to melt double- stranded DNA (dsDNA). This characteristic of the E. coli polymerase made PCR a challenging and laborious technique because fresh polymerase had to be added before each elongation step.
The isolation of DNA polymerase from the thermophilic bacterium Thermus aquaticus greatly advanced PCR (Chien et al. 1976). Because T. aquaticus naturally flourishes in high- temperature environments, its DNA polymerase, often referred to as Taq polymerase, is naturally thermostable. The thermostability of Taq polymerase enables it to withstand the high temperatures necessary for PCR reactions. Taq polymerase is considered a highly processive polymerase because it can catalyze the addition of approximately 60 nucleotides/sec at 70°C (Innis et al. 1988).
Although the Taq polymerase is thermostable and highly processive, it has limitations in certain PCR applications. Because it is catalytically active at room temperature, it may elongate PCR primers that bind nonspecifically to DNA during the reaction setup. To prevent this nonspecific amplification during experimental preparations, reactions may be prepared on ice or the polymerase can be added separately after the initial denaturation step. Alternatively, the nonspecific amplification associated with Taq polymerase can be circumvented through the use of engineered hot-start enzymes.
Hot-Start DNA Polymerase
Special DNA polymerases that remain in an inactive state at ambient temperature have been engineered to prevent nonspecific amplification during PCR assay preparation. DNA polymerases dependent on high-temperature incubation for activation are termed "hot-start DNA polymerases." These DNA polymerases have blocking molecules or inhibitory antibodies covalently linked to the polymerase. The first step of PCR with a hot-start polymerase requires a high-temperature incubation to irreversibly denature the inhibitory molecule and release the active enzyme. This incubation typically ranges from 1 to 10 min at approximately 95°C.
Proofreading DNA Polymerase
Taq has an error rate of approximately 1 in every 9,000 base pairs (Tindall and Kunkel 1988). The limited fidelity of Taq is due to its lack of 3' to 5' exonuclease activity, or the ability of the enzyme to remove the terminal, mismatched nucleotide from a DNA strand by cleaving the phosphodiester bond between bases.
In 1991, scientists described a thermostable DNA polymerase isolated from Pyrococcus furiosus that possesses 3' to 5' exonuclease proofreading capabilities (Lundberg et al. 1991). This proofreading enzyme, referred to as Pfu DNA polymerase, removes the misincorporated base and allows extension to continue. With an error rate of 1 in 1.3 million bases, the DNA replication fidelity of Pfu DNA polymerase is significantly higher than that of Taq polymerase. Proofreading enzymes are often used when precise sequences are extremely important, such as in expression and mutational analyses and in cloning procedures.
Unlike Taq polymerase, Pfu DNA polymerase and other conventional proofreading enzymes have reduced processivity and require much longer PCR extension times. In recent years, researchers have engineered novel proofreading DNA polymerases that have greatly increased enzyme processivity. For example, the Bio-Rad iProof™ high-fidelity DNA polymerase comprises a unique proofreading enzyme fused to a dsDNA binding protein that enhances processivity. This allows for the generation of long templates with an accuracy and speed previously unattainable with a single enzyme.
Following PCR amplification, certain downstream reactions require a PCR purification step to remove reaction mixture components (including salts, enzymes, primers, primer-dimers, and unincorporated dNTPs) from the PCR product. The purity of the DNA template is the most critical factor in PCR-based DNA sequencing because the presence of contaminants decreases the quality of the sequence reads significantly. Contaminated samples exhibit a high background and can lead to misinterpretation of sequencing data.
A PCR product can be purified using a spin column format in which the amplified DNA fragment binds to a chromatographic support based on the pH and ionic strength of the solution. DNA bound to the column is subjected to low- and high-stringency washes to remove contaminants. The purified nucleic acid is eluted with a low ionic strength buffer. The amplified DNA fragment is then ready for most downstream applications such as digestion by restriction enzymes, ligation into a vector or labeling.
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