Optimization of a real-time assay is covered in more detail in Chapters 1 and 3, however it cannot be over-stressed how important the careful optimization of all reaction components and conditions is. Primer and probe concentrations should be optimized to give the earliest signal for a specific amplicon, this ultimately equates to the sensitivity of the reaction. The annealing temperature for any given primer set, or primer/probe combination can be calculated by any number of different means. These calculated temperatures are not always the temperatures at which the best performance is achieved. Standard PCR on a gradient block PCR machine can be used to check the specificity of real-time primers. To check the specificity of a primer/probe combination then a gradient on a real-time PCR machine will allow visualization of the fluorescence signal from the probe. The melting curve from a SYBR® Green real-time reaction gives a reasonable indication as to the number of products amplified in a reaction. However, as the amplicon 'melts' at a range of temperatures along its length until completely single stranded, these peaks are quite broad. Products very similar in size may not be distinguishable by this approach.
Magnesium chloride concentration affects the activity of the polymerase in the reaction. Polymerases from different manufacturers will all require different magnesium chloride concentrations to enable them to perform maximally. The best indicator for optimization is the PCR efficiency and reaction reproducibility. The PCR efficiency is taken from the number of PCR cycles between log dilutions of a standard template and was discussed earlier. Most manufacturers supply reagents with an optimized buffer. This should perform well for the majority of amplicons. However, magnesium chloride concentration should be optimized for each reaction by amplifying a dilution series at a range of magnesium chloride concentrations. Comparison of the PCR efficiencies will enable the investigator to select the optimum concentration for each assay they design. Figure 11.4 shows a magnesium chloride optimization experiment for an mtDNA real-time assay in the ND1 region. Magnesium chloride concentrations of 1.5 mM, 2.5 mM, 3.0 mM, 3.5 mM, 4.0 mM and 4.5 mM are shown in Figure 11.4 parts A to F respectively. As mentioned previously investigators should aim to have reactions with a slope as close to -3.3 as possible. An important issue is the consistency of efficiency. An efficiency of 96% on one run and then 65% on a subsequent run highlights an assay that is not well optimized.
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