Gradient Polymerase Chain Reaction to Determine the Optimum Annealing Temperature

Gradient polymerase chain reaction is a technique to optimize the annealing temperature at which primers — sequence-specific, single-stranded DNA — bind to the complementary sites on template DNA.

Take a mix containing forward and reverse primers, dNTPs, and thermostable DNA polymerase in an appropriate buffer. Add the template DNA and transfer the constituents into a PCR plate.

Place the plate in a gradient thermal cycler with a specialized heating block. This establishes a linear gradient of annealing temperature between the range of 55 °C to 65 °C, with a consistent increment in temperature throughout the plate.

The reaction heats the plate at a high temperature, denaturing the double-stranded DNA into single strands and activating the DNA polymerase. Later, the temperature is lowered according to the programmed values in each well, facilitating primer annealing.

At below-optimum annealing temperature, the primers bind non-specifically to the denatured DNA, leading to non-specific amplification during the PCR reaction. At above-optimum temperature, insufficient quantities of the primers bind to the DNA.

At optimum temperature, the primers bind specifically to the target DNA. Then, the temperature slightly increases, allowing the polymerase to extend the primers using dNTPs. These repetitive denaturation, annealing, and extension cycles lead to DNA amplification.

Analyze the product in each well. The wells with non-optimum annealing temperatures show less or non-specific amplification.

An optimum annealing temperature produces the maximum specific amplification of DNA.