Producing high yields of good-quality PCR products requires a complex combination of optimal chemistry, primer design, template quality, and cycling parameters. The details of these reaction conditions change for every PCR product, and suboptimal reagent concentrations and annealing times lead to sequence errors, incorrect product sizes, nonspecific products, or a lack of product. Therefore, researchers must optimize their PCR reagents and conditions for every new template and primer set.
The unique features of every DNA template change the PCR chemistry. Researchers should modify their starting template concentration based on the DNA composition and type. For example, a PCR using a genomic DNA template requires a higher template concentration compared to one with a plasmid DNA template. Additionally, the DNA concentration may need to be altered to accommodate different DNA polymerases.
Once a researcher determines the ideal template concentration, they should optimize their reaction’s amplification efficiency by testing the other reaction components. For example, difficult DNA templates and samples with enzyme inhibitors require adjusted polymerase concentrations, and primers comprised of degenerate bases should be used at higher concentrations. However, researchers must be careful when altering the template, primer, or polymerase concentrations because increased amounts can cause nonspecific amplification while decreased amounts can reduce product yield.
It is important to also optimize other reaction components, including MgCl2, dNTPs, and the buffer. Magnesium interacts with dNTPs, primers, the template, and the polymerase to facilitate dNTP polymerization. Too little magnesium reduces polymerase activity, while too much causes replication errors. An optimized concentration maximizes yield and retains product specificity. dNTPs are typically added in equimolar amounts for standard PCR reactions. However, repeated freezing and thawing can lead to degradation and concentration imbalances in dNTP mixes. Researchers can increase the dNTP concentration if using more magnesium in the reaction or when amplifying long fragments. Alternatively, a lower dNTP concentration may improve the fidelity of non-proofreading polymerases. Finally, to provide a suitable environment for the reaction chemistry and to discourage mismatched base-pairing, researchers should explore buffers with different compositions and pH values to find the ideal formulation.
PCR optimization requires careful experimental design to test reagent performance in reaction specificity, sensitivity, efficiency, and repeatability. To do this, researchers make serial dilutions of their PCR reagents, changing one variable at a time to find the optimal concentrations. Additionally, different annealing temperatures beyond the one calculated from the primer composition may also enhance reaction performance.
With so many variables, researchers often struggle with or skip PCR optimization. Furthermore, altering component concentrations is difficult when using commercial PCR master mixes. Researchers can easily find their ideal set of reaction conditions to make custom master mixes using the Promega PCR Optimization Kit. This kit contains reagents designed to help researchers identify their ideal master mix formulation through a simple optimization process. The kit contains MgCl2, GoTaq® MDx Hot Start Polymerase, and a spectrum of PCR buffers for different amplification needs, such as endpoint, multiplex, and real-time reactions and GC-rich and inhibitor-resistant targets. To use the kit, researchers first set up a series of PCR reactions with each buffer and titrated magnesium concentrations to find the buffer type and MgCl2 concentration that produces the best results. Next, they perform another set of reactions to identify the optimal annealing temperature for the conditions they identified in step one. Finally, researchers refine the polymerase and magnesium concentrations in a third set of reactions.
Promega offers custom amplification reagents, including buffers, MgCl2, dNTPs, DNA polymerases, and reverse transcriptases, that are manufactured according to rigorous quality standards. Because Promega is the primary manufacturer, customers collaborate directly with the scientists who design and manufacture their products. In addition to individual reagents, the scientists at Promega produce custom PCR master mixes from formulations already in use or based on the results from the PCR Optimization Kit. Reagent customization and bespoke master mixes give researchers the confidence that their PCRs will efficiently produce good-quality products at high yields.
- E.V. Pelt-Verkuil et al., Principles and Technical Aspects of PCR Amplification, Dordrecht: Springer, 2008.
- T.C. Lorenz, “Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies,” J Vis Exp, 3998, 2012.