Table 11.3 Positive and Negative Effectors of a PCR Reaction To Enhance this Parameter Manipulate One or More of These Components Fidelity and Enzyme Select an enzyme with potent 3-5 Exonuclase activity Primer design Include mismatches at 3 end. which can help discriminate against homologous equences such as pseudogenes Enzyme selection can enhance this effect. with Taq polymerase, relative amplification efficiencies with 3 -terminal mismatches is greater for A: G and C: C than for other nucleotide pairs(Kwok et al., 1995) Use longer primers(refer to section"What Are the Steps to Good Primer Design? Primers less than 15 nucleotides do not give enough specificity from a statistical f view PCR cycling condition Increase annealing temperature Reduce cycle segment time(denaturation Healing, etc. ) Lower cycling number Reaction chemistry Apply a hot start strategy(Erlich, Gelfand, Check that concentration and pH of dNTP olution(s)is correct. Decrease primer concentration. Template Confrim that template is intact, not nicked, and free of contaminants and inhibitors opy numhpresence of sufficient starting Confirm the Method of analy Minimize contamination and handling errors: use an automated analysis systen Use sufficient sample number to enable reliable statistical analysis Check for erroneous manipulation (pipetting errors, etc. eal tice Use a positive displacement pipet Use a separate room to set up experiments. Use UNG and dUTP (Longo, Berninger, and Hartley, 1990) Cycler Check that the temperature pr consistent at every position in the he block Decrease ramp time. Check for tight fit between reaction vessels and heating block Efficiency of doubling/cycle Reaction ncrease concentration of dnTPs and Use minimal concentrations of DMso DME, formamide, SDS, gelatin, glycerol (see Table 11.7) PCRPCR 297 Table 11.3 Positive and Negative Effectors of a PCR Reaction To Enhance This Parameter Manipulate One or More of These Components Fidelity and specificity Enzyme Select an enzyme with potent 3¢–5¢ Exonuclase activity. Primer design Include mismatches at 3¢ end, which can help discriminate against homologous sequences such as pseudogenes. Enzyme selection can enhance this effect. With Taq polymerase, relative amplification efficiencies with 3¢-terminal mismatches is greater for A :G and C :C than for other nucleotide pairs (Kwok et al., 1995). Use longer primers (refer to section “What Are the Steps to Good Primer Design?”. Primers less than 15 nucleotides do not give enough specificity from a statistical point of view. PCR cycling condition Increase annealing temperature. Reduce cycle segment time (denaturation, annealing, etc.). Lower cycling number. Reaction chemistry Decrease [Mg2+ ]. Apply a hot start strategy (Erlich, Gelfand, and Sninsky, 1991). Check that concentration and pH of dNTP solution(s) is correct. Decrease primer concentration. Template Confrim that template is intact, not nicked, and free of contaminants and inhibitors. Confirm the presence of sufficient starting copy number. Method of analysis Minimize contamination and handling errors; use an automated analysis system. Use sufficient sample number to enable reliable statistical analysis. Check for erroneous manipulation (pipetting errors, etc.). Clean lab practice Use a positive displacement pipette. Use a separate room to set up experiments. Wear gloves. Use UNG and dUTP (Longo, Berninger, and Hartley, 1990). Cycler Check that the temperature profile is consistent at every position in the heating block. Decrease ramp time. Check for tight fit between reaction vessels and heating block. Efficiency of doubling/cycle Reaction Increase concentration of dNTPs and enzymes. Use minimal concentrations of DMSO, DMF, formamide, SDS, gelatin, glycerol (see Table 11.7)