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. The