Figure 1 shows an agarose gel with the 12 samples that were loaded across the block. Clearly, the best conditions are found in well 10 of the cycler where the temperature was Figure 1: Experimental determination of optimal annealing temperature: The calculated primer annealing temperature was The ribosomal spacer region of mycoplasms from H9 cell cultures was amplified.
The following test parameters were selected: denaturation 10 s, annealing 15 s, elongation 20 s, Taq-Polymerase 0. In Figure 2, the amplification shown in Figure 1 performed again, this time under optimized temperature conditions. In this experiment, the universal block was set to a uniform temperature in the annealing phase. The following test parameters were used: denaturation 94,10 s, annealing Figure 2 shows the agarose gel with 12 samples loaded across the block.
Figure 2: Amplification shown in Figure 1 performed under optimized temperature conditions. The outstanding temperature homogeneity of the block ensures reproducible PCR results. For example, in the presence of 0. The gradient function of the Mastercycler is not restricted to the annealing temperature. The gradient function can also be used for optimization of the denaturation step, or the elongation temperature.
However, special target DNAs require a different denaturation temperature to achieve an optimal result. The gradient would determine the lowest possible denaturation temperature providing high yield of amplified DNA.
The exposure of the Taq Polymerase to unnecessary high temperatures would decrease the enzyme activity over the PCR run, which would have to be compensated by using more enzyme. An initial denaturation step of 1 to 5 minutes 3 - 10 could further optimize the reaction. Harmful nucleases in the sample will be inactivated, ensuring the complete denaturation of complex templates such as genomic DNA. In special cases the primer annealing temperature may need to be raised as high as the extension temperature.
In fact, high-temperature annealing should result in enhanced specificity, because the hybridization of the primer to the template DNA occurs under more stringent conditions. Combining primer annealing and primer extension steps results in a two-step PCR protocol. Again, the optimal temperature can easily be determined with the gradient function of the Mastercycler. Tel: , ext.
Naturally, decades of improvements see more and more stable PCR reagent formulations — increasing fidelity, specificity and speed performance of enzymes. For example, difficult templates such as GC-rich sequences require higher temperature. Higher denaturation temperature can also lead to higher specificity in PCR amplification 3.
Things become more complicated when choosing the right annealing temperature. The closest reference one has is the melting temperatures TM of the forward and reverse primers.
After all, you must choose only one temperature to perform the annealing with. The bigger the difference, the more bias you have towards one primer or the other. Unfortunately, TM can change with reagent concentration, pH and salt concentration. Hence, TM remains a theoretical value that can only provide a rough guideline to determine the actual annealing temperature TA. Using higher TA results in higher specificity but may affect the yield as it means less binding more stringent binding condition.
Thus, finding the most efficient and specific TA is often a trial and error endeavor. What is gradient technology? It is a technology where instead of giving the same temperature throughout the entire thermal block of a thermal cycler, each of the columns or rows in the block is made to have different temperatures. This means that you can either choose to run repeated PCRs testing different temperatures — each about an hour long — to find the right TA. Or, you can choose to use a thermal cycler equipped with gradient function.
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