A number of different factors can significantly impact on PCR sensitivity and specificity, including (1) oligonucleotide primer design, (2) PCR cycling parameters (number of cycles, cycle times, and temperatures), and (3) the composition of the PCR mixture (Mg2+ concentration). For most PCR applications, the most critical parameter is the annealing temperature for the oligonucleotide primers employed. The maximum annealing temperature is determined by the primer with the lowest Tm. Exceeding this Tm by more than several degrees will diminish the ability of the oligonucleotide primer to anneal to the target sequence and could result in the failure to produce the product of interest. If an annealing temperature equal to Tm of the oligonucleotide primers fails to produce the desired product, then it might be necessary to lower the annealing temperature further. If the desired amplicon is produced but the level of background products is high, then the annealing temperature should be increased. Salt concentrations also affect several aspects of PCR. Mg2+ concentration can affect oligonu-cleotide primer annealing to the target sequence, the Tm of oligonucleotide-template complexes, and enzyme activity and fidelity. Taq polymerase requires free Mg2+ for activity. Therefore, sufficient MgCl2 must be included in the PCR mixture to provide sufficient Mg2+ for the enzyme after some of the cation is lost to chelation by the oligonucleotide primers and the template DNA. The concentration of other salts can affect the PCR as well (including KCl). However, optimization of most
PCR applications can be achieved through modification of Mg2+ concentration.
Complete optimization of the reaction conditions might require several adjustments to the annealing temperature, PCR cycle parameters, and salt concentrations. PCR optimization kits are commercially available. These kits include various PCR buffers and enable modification of various components of the PCR mixture, enabling optimization in less time and with fewer steps. For instance, the PCR Optimization Kit (from Roche Applied Science, http://www.roche-applied-science.com) contains a set of 16 buffers facilitating the simultaneous variation of pH and MgCl2 concentration, as well as PCR mixture additives for further optimization (DMSO, gelatin, and glycerol).
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