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Using Gradient PCR to determine the optimum annealing temperature.

Problems with the amplification of a specific DNA fragment using the PCR* are an everyday occurrence in the lab. For example, unspecific secondary bands may form after the PCR reaction, which hinder, or even prevent, further analyses (cycle sequencing, mutation detection, etc.) or an unequivocal assessment of the PCR result. In such cases, PCR conditions must be optimized. This is normally achieved by titrating the magnesium-, template-, primer-, dNTP- and Taq polymerase concentration, Hot Start PCR, Touch-down PCR, adding detergents, reducing the PCR cycles or by gradually increasing the annealing temperature. Fig. 1: Experimental determinati on of optimal annealing temperature: The calculated primer annealing temperature was 56.5C, the actual annealing temperature is 63.5C. The ribosomal spacer region of mycoplasms from H9 cell cultures was amplified. Using the gradient function of the universal block, a gradient of 53 to 67C was set. The following test parameters were selected: denaturation 10 s, annealing 15 s, elongation 20 s, amount of Taq-polymerase 0.75 units; duration of entire experiment: <1 h. Fig. 2: Amplification shown in fig. 1 performed under optimized temperature conditions. In this experiment, the universal block was set to a uniform temperature in the annealing phase. The outstanding temperature homogeneity of the block ensures reproducible PCR results. The selection of the annealing temperature is possibly the most critical component for optimizing the spec ificity of a PCR reaction. In most cases, this temperature must be empirically tested. The PCR is normally started at 5C below the calculated temperature of the primer melting point (Tm). However, the possible formation of unspecific secondary bands shows that the optimum temperature is often much higher than the calculated temperature (>12C).

Further PCR reactions with gradually increasing temperatures are required until the most stringent conditions have been found. When a standard PCR cycler is used, this method is the most time-intensive optimization strategy.

The Mastercycler gradient now enables rapid testing of the optimum temperature conditions on one block and in one experiment. During the PCR, a temperature gradient, which can be programmed between 1C and 20C, is built up across the thermoblock. This allows the most stringent parameters for every primer set to be calculated with the aid of only one single PCR reaction.

If the gradient function is not used, the block offers precise homogeneity, thus ensuring first-class reproducible results.

Example for Gradient PCR Program
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