Improve Yield and Specificity of Your PCR Using the 2D-Gradient Function

What is 2D-Gradient function?

As mentioned above PCR optimization, typically by finding the right T_A using a gradient function, is an established technique. Optimization of the denaturation temperature (T_D) is less commonly done and typically limited to applications dealing with long or GC-rich DNA templates as well as mastermixes with high salt content. In addition, optimizing the denaturation temperature is usually not a focus because the impact of the annealing temperature optimization is considered bigger. Still, an optimized denaturation temperature can lead to a higher yield and thus should be kept in mind, especially for applications requiring large amounts of DNA such as cloning or sequencing. Testing both, a range of annealing and denaturation temperatures, however, is a laborious endeavor.

A 2D-Gradient speeds up simultaneous T_A and T_D optimization significantly by providing two temperature gradients; one along the x-axis and one along the y-axis of the PCR cyclers’ thermoblock (Figure 1). This enables quick testing of 96 T_A and T_D combinations in one run to screen for the optimal temperature combination to eliminate unspecific signals and increase product yield.

Why is an optimization of annealing and denaturation temperature beneficial?

The optimization of both temperatures offers improved specificity and yield (Figure 2) resulting in several advantages. The optimal temperature combination can be selected according to the needs of your workflow.

Improved specificity:

• Clearer gel bands for publications
• Specific target DNA for post-PCR processing
• Less false-positive (unspecific) results

Improved yield:

• Faster PCR-protocol possible -> save time
• Lower reaction volume possible -> save money
• Can work with less template -> save money & work
• More DNA for downstream applications possible -> efficient workflow
• Get sufficient yield in difficult assays -> process stability

Advantages of 2D- vs. 1D-Gradient

As described above, the 2D-Gradient function enables testing of 96 temperature combinations in a single PCR run. The optimization of ß-actin gene amplification shown in Figure 2 may have required eight PCR runs when using a thermal cycler with a 1D-Gradient function. Thus, the 2D-Gradient function helps to save time labor, reagents, consumables, and energy for temperature optimization. Furthermore, the results are more comparable as only a single mastermix, PCR plate, and sealing are needed when using the 2D-Gradient function (see Figure 3).

All variants of the Mastercycler X50 family are equipped with the 2D-Gradient function.