To Enhance this Manipulate One or More of These Components electrophoresis. When hybridization obes are used, primer-dimer formation will not mask the authentic product, even after 40 cycles. This is not true for reen o Nested PCR or extra manipulation may be eeded for other non-real-time pcr “Hot"” nested Pcr is one such example that elegantly ombines the qualities of nested PCR ith the high resolution of page (Jackson, Hayden, and Quirke, 1991) Control Include ositive and negative controls when the target is not detected, one can conclude that target was below 100 copies, etc, which makes the data more meaningful than just saying it was not Lab setup Clean lab No contamination Experimental design primer desig enes is a concern, design the primer to create mismatches at the 3 end using the most heterogeneous fidelity, but one needs to be aware when high fidelity has to be considered. During planning, one should also consider the many ways a PCR reaction can be manipulated to achieve a given end as discussed throughout this chapter The data in Table 11. 4 are provided to highlight the biochemi- cal properties of common PCR-related enzymes and help you develop a selection strategy. For a comprehensive comparisor of thermostable DNa polymerases, see Perler, Kumar, and Kong(1996), Innis et al.(1999), and Hogrefe(2000). However, biochemical data and logic can't always predict the most appro- priate enzyme for PCR; experimentation might still be required to determine which enzyme works best. Abu Al-Soud and Rad- strom(1998) demonstrate that contaminants inhibitory to PCR vary with the sample source, and that experimentation is required to determine which thermostable dna polymerase will produ successful PCR. A second illustration of the difficulty in predict ing success based on enzymatic properties are the Archae DNA polymerases, which have not become premiere PCR enzymes despite their extreme thermostability and good proofreading activity.Table 11.3 (Continued) To Enhance This Parameter Manipulate One or More of These Components electrophoresis. When hybridization probes are used, primer-dimer formation will not mask the authentic product, even after 40 cycles. This is not true for SYBR® Green or Amplifluor. Nested PCR or extra manipulation may be needed for other non-real-time PCR based techniques. “Hot” nested PCR is one such example that elegantly combines the qualities of nested PCR with the high resolution of PAGE (Jackson, Hayden, and Quirke, 1991). Control Include positive and negative controls; when the target is not detected, one can conclude that target was below 100 copies, etc., which makes the data more meaningful than just saying it was not detected. Lab setup Clean lab. No contamination. Experimental design Check primer design. If amplifying related genes is a concern, design the primer to create mismatches at the 3¢ end using the most heterogeneous sequence region. fidelity, but one needs to be aware when high fidelity has to be considered. During planning, one should also consider the many ways a PCR reaction can be manipulated to achieve a given end, as discussed throughout this chapter. The data in Table 11.4 are provided to highlight the biochemi￾cal properties of common PCR-related enzymes and help you develop a selection strategy. For a comprehensive comparison of thermostable DNA polymerases, see Perler, Kumar, and Kong (1996), Innis et al. (1999), and Hogrefe (2000). However, biochemical data and logic can’t always predict the most appro￾priate enzyme for PCR; experimentation might still be required to determine which enzyme works best. Abu Al-Soud and Rad￾strom (1998) demonstrate that contaminants inhibitory to PCR vary with the sample source, and that experimentation is required to determine which thermostable DNA polymerase will produce successful PCR. A second illustration of the difficulty in predict￾ing success based on enzymatic properties are the Archae DNA polymerases, which have not become premiere PCR enzymes despite their extreme thermostability and good proofreading activity