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,
, 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:
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.
for Gradient PCR Program
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