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Temperature gradient vs. homogeneity: How can gradient PCR optimize your qPCR assay?

By: Roche Life Science

Posted: March 21, 2016 | Lab Life - Real-Time PCR

Optimization of qPCR conditions is essential for maximizing the efficacy and reproducibility of your assay. While there are numerous variables at play in your qPCR experiment, the most critical component for efficiency and robustness is optimization of your primers. Indeed, when designing new primers, it is critical to determine the proper annealing temperatures.

While many primers are variably effective at a range of annealing temperatures, there are instances where even a difference of 2-4 degrees Celsius could result in changing a target amplicon from undetectable to reliably robust. However, while PCR is typically started at 5 degrees Celsius below the calculated primer melting point (Tm), in most cases the optimal annealing temperature must be empirically tested.

Gradient PCR to determine optimal annealing temperatures
Testing your reaction at a fixed primer concentration over a range of annealing temperatures is the most effective way to determine the optimal annealing temperature of your primers in the least amount of steps. This is most efficiently performed with the use of a qPCR instrument with a temperature or thermal gradient block, also known as gradient PCR. These instruments allow the use of a range of temperatures in different regions of the block. Ideally, this block should have high reproducibility and uniformity of the gradient temperatures from run to run. The initial temperature range chosen is the lowest and highest that might be appropriate for a given primer set and generally occurs between 52 to 70 degrees Celsius.

When evaluating a new set of primers for a given target amplicon, the first step is to perform the gradient PCR using a fixed concentration of primers and template. Choose the appropriate annealing temperature range, such as 52-70 degrees Celsius. Depending on your thermocycler instrument, the locations of the annealing temperatures may be column- or row-based. For instance, column 1 is at 52 degrees Celsius, column 2 is at 54 degrees Celsius, column 3 is at 56 degrees Celsius, and so forth. Your PCR mastermix should include the same concentrations of your remaining ingredients (dNTPs, Mg, template, enzyme, and primer). This ensures that each reaction will only vary by the temperature with all other variables remaining the same.

Once your reactions are complete, you can run the PCR products on an agarose gel to see which annealing temperature produced the appropriately sized fragment as a single band. You may want to verify the fragment is indeed the correct product by sequencing or restriction enzyme digest. If multiple annealing temperatures seem to provide the appropriate target, choose the highest of these temperatures, as this should result in enhanced specificity due to hybridization of the primer to the template DNA at more stringent conditions.

Additional functions for gradient PCR
While the most well-utilized function for gradient PCR is optimization of annealing temperature, it can also be used for optimization of denaturation or elongation should your assay require it. While the most common temperature range for DNA denaturation is 94 to 96 degrees Celsius, there are certain DNA target sequences that optimally denature at temperatures outside of this range for providing higher yields of amplified DNA. This can be accomplished by gradient PCR with testing of temperature ranges from 90 to 99 degrees Celsius to find the optimal denaturation temperature. Additionally, gradient PCR can be utilized for optimizing two-step PCR protocols, combining primer annealing and extension steps. In these assays, the annealing-extension temperature may be somewhere in the range of 60 to 72 degrees Celsius, while extension in single-step reactions generally occurs at 72 degrees Celsius.

In summary, gradient PCR can help you optimize annealing, extension, or denaturation temperatures in a single run. Thermal gradient blocks can help you determine the optimal annealing temperature for multiple primer sets or even to perform various reactions at different annealing temperatures at the same time.

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