In a polymerase chain reaction (PCR), DNA molecules are copied, or amplified, using an enzyme called DNA polymerase. During a PCR reaction, strands of DNA are separated from each other and bound by short, specifically-designed DNA sequences called primers that provide a starting point for the DNA polymerase to begin the copying process. Polymerases extend the new DNA strand by adding nucleotides, the building blocks of DNA, to the end of the primer sequence.
Many versions of DNA polymerase exist, each with a specific set of functional characteristics that can affect the success of a PCR reaction. Here are some tips for selecting the best enzyme for your experiments based on the different properties of DNA polymerases.
1.Balancing fidelity and yield
To prevent mistakes while copying DNA, some polymerases have a “proofreading” function, which can remove nucleotides that were incorrectly added to the growing DNA strand. These high-fidelity, or “HiFi,” polymerases are ideal for cloning projects or other applications that require accuracy.
Since the enhanced accuracy of HiFi polymerases increases cost and decreases the reaction speed and yield, a non-proofreading polymerase may be more suitable for standard or large-scale PCR experiments.
2.Increasing specificity
Usually obtained through careful primer design, specificity refers to amplifying only the intended DNA sequence. Certain polymerases enhance specificity by preventing the enzyme from copying DNA molecules non-selectively at low temperatures during reaction set-up.
These hot-start polymerases reduce contamination and enhance product yield. Although they lack proofreading activity, hot-start polymerases are particularly useful when amplifying DNA from complex mixtures or low amounts of starting material, or when using multiple sets of primers for multiplex PCR.
3.Processivity and reaction speed
The processivity of a polymerase describes how many new nucleotides the enzyme can add to the growing DNA strand before falling off. Polymerases with higher processivity tend to be faster and result in a higher overall yield. Typical polymerases copy anywhere from 1-4 kb of DNA sequence per minute.
4.Master mixes
PCR reactions require many factors in addition to DNA polymerase, such as Mg2+, salt, and free nucleotides. Many polymerases can be purchased as a master mix that includes all required components at pre-optimized concentrations in a single buffer. Though more expensive, master mixes are convenient and reduce the chance of pipetting errors during PCR set-up.
For difficult reactions, it may be necessary to troubleshoot the concentrations of individual reaction components.
5.Experiment-specific considerations
The design of the PCR experiment can dictate which polymerase to use, based on the desired balance of specificity, sensitivity, accuracy, and product abundance.
For example, high-fidelity polymerases prevent mistakes from accumulating when amplifying long DNA molecules, and special long-range polymerases are best for amplifying exceptionally long molecules.
For genotyping SNPs or amplifying from complex starting material such as genomic DNA, a polymerase with higher specificity may be important.
Stellar Scientific offers an array of DNA polymerases, including standard Taq for maximal reaction speed and affordability, hot-start and high-fidelity options for increased specificity and accuracy, and long-range polymerases designed to amplify difficult DNA templates up to 35kb in length