武汉大学生命科学学院：《分子生物学》（英文版）11 PCR

days. One-step RT-PCR is done using the same RNa samples, and PCr products are analyzed by polyacrylamide gel elec trophoresis (PAGE). For some unknown reason, the second experiment shows different quantitative data. Which data ar correct? Without a sufficient number of samples to calculate stan- dard deviation, one cannot make any quantitative analysis For quantitative PCR, the sample size has to be large enough and the standard curve must show that PCR was linear within the range one is examining. To do this, serial dilution of a positive control must be run simultaneously, and the test samples have to fall within this range of amplification. Minimums of three to four samples are required for reliable statistical analysis of the data. It is also a good idea to generate enough cDNA to run multiple experiments to reduce error due to differences in the cDNA syn hesis step. The positive control must also be properly stored so hat loss or damage of DNa does not generate false negative results High-tech, automated PCR synthesis and detection systems are useless if the sample preparation destroys the mRNA, co-purifies PCR inhibitors, or the PCr primer design amplifies genomic DNA. Your results will only be as good as the weakest parameter in your PCR strategy Manipulate the Reaction to Meet Your Needs Table 11.3 describes positive and negative effectors of the PCr reaction. These data can help you plan your experiment or modify our strategy if your results aren't satisfacto DEVELOPINGA PCR STRATEGY THE EXPERIMENTAL STAGE What Are the Practical Criteria for Evaluating a dNA Polymerase for Use in PCR An appreciation of what your research objective requires from a PCr product should be central to your selection of a ther mostable dNa polymerase. Were you planning to identify a rare mutation in a heterogeneous population as in allelic polymor- phisms(Frohman, Dush, and Martin, 1988)? As the copy number gets smaller (less than 10), the need for high-fidelity enzyme or enzyme mixes increases, as discussed below. In contrast, if you're screening a batch of transgenic mice for the presence or absence of a marker gene via Southern hybridization, enzyme fidelity might not be as crucial. Most applications do not require high 296 Aoyagi

296 Aoyagi days. One-step RT-PCR is done using the same RNA samples, and PCR products are analyzed by polyacrylamide gel elec￾trophoresis (PAGE). For some unknown reason, the second experiment shows different quantitative data. Which data are correct? Without a sufficient number of samples to calculate stan￾dard deviation, one cannot make any quantitative analysis. For quantitative PCR, the sample size has to be large enough and the standard curve must show that PCR was linear within the range one is examining. To do this, serial dilution of a positive control must be run simultaneously, and the test samples have to fall within this range of amplification. Minimums of three to four samples are required for reliable statistical analysis of the data. It is also a good idea to generate enough cDNA to run multiple experiments to reduce error due to differences in the cDNA syn￾thesis step. The positive control must also be properly stored so that loss or damage of DNA does not generate false negative results. High-tech, automated PCR synthesis and detection systems are useless if the sample preparation destroys the mRNA, co-purifies PCR inhibitors, or the PCR primer design amplifies genomic DNA. Your results will only be as good as the weakest parameter in your PCR strategy. Manipulate the Reaction to Meet Your Needs Table 11.3 describes positive and negative effectors of the PCR reaction. These data can help you plan your experiment or modify your strategy if your results aren’t satisfactory. DEVELOPING A PCR STRATEGY: THE EXPERIMENTAL STAGE What Are the Practical Criteria for Evaluating a DNA Polymerase for Use in PCR? An appreciation of what your research objective requires from a PCR product should be central to your selection of a ther￾mostable DNA polymerase. Were you planning to identify a rare mutation in a heterogeneous population as in allelic polymor￾phisms (Frohman, Dush, and Martin, 1988)? As the copy number gets smaller (less than 10), the need for high-fidelity enzyme or enzyme mixes increases, as discussed below. In contrast, if you’re screening a batch of transgenic mice for the presence or absence of a marker gene via Southern hybridization, enzyme fidelity might not be as crucial. Most applications do not require high

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) PCR

PCR 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)

Table 1 1.3(Continued) To Enhance This Manipulate One or More of These Componen infirm that template is unnicked, free of contaminants and inhibi Use a smaller size template DNA (get more molecules per pg of input template, and less complexity for primer annealing). For example, PCR product vs genomic DNA Decrease amplicon size. Taq>Pfu, >>Stoffel fragment. Decrease cycling time or use a shuttle profile( Cha et al., 1992) Decrease the size of the tube Check for tight fit between reaction vessels and heating block. Decrease ramp time Use forward and reverse primers that have milar length and gC content. nfirm that primers do not form primer dimer or hairpin structure Reproducibility Ensure that template is clean and intact onfirm presence of sufficient starting template and sufficient sample numbe for statistical analysis. Use the same lots of primer and buffers between experiments. Store enzyme in small aliquots. for presence of contaminating template and inhibitors to PCR reaction. trols Include positive and negative controls with all experiment Cyclin Use a hot-start strategy( Kellogg et al Use the same cycler between experiments. Quantitative Confirm the quantity of the template Confirm template preparation is clean. Investigate for presence of contaminating template and inhibitors to PCR reaction Experimental design Include triplicate or quadruplicate samples Use a statistically sufficient number of sam repare a standard curve to demonstrate the range over which PCR product yield provides a reliable measure of the Robust: Confirm that chemistry, primer design, tubes, thermal cycler, and other factors are optimized. Confirm the analytical methods accuracy/resoluton. Is it accurate during the exponential phase of PCR? 298 Aoyagi

298 Aoyagi Table 11.3 (Continued) To Enhance This Parameter Manipulate One or More of These Components Template Confirm that template is unnicked, free of contaminants and inhibitors. Use a smaller size template DNA (get more molecules per pg of input template, and less complexity for primer annealing). For example, PCR product vs. genomic DNA. Decrease amplicon size. Enzymes Taq > Pfu, >>Stoffel fragment. Cycling Decrease cycling time or use a shuttle profile (Cha et al., 1992). Decrease the size of the reaction tube. Check for tight fit between reaction vessels and heating block. Cycler Decrease ramp time. Primer design Use forward and reverse primers that have similar length and GC content. Confirm that primers do not form primer￾dimer or hairpin structure. Reproducibility Sample Ensure that template is clean and intact. Confirm presence of sufficient starting template and sufficient sample number for statistical analysis. Reagents Use the same lots of primer and buffers between experiments. Store enzyme in small aliquots. Investigate for presence of contaminating template and inhibitors to PCR reaction. Controls Include positive and negative controls with all experiments. Cycling Use a hot-start strategy (Kellogg et al., 1994). Use the same cycler between experiments. Quantitative Template Confirm the quantity of the template. Confirm template preparation is clean. Investigate for presence of contaminating template and inhibitors to PCR reaction. Experimental design Include triplicate or quadruplicate samples. Use a statistically sufficient number of samples. Prepare a standard curve to demonstrate the range over which PCR product yield provides a reliable measure of the template input. Robust: Confirm that chemistry, primer design, tubes, thermal cycler, and other factors are optimized. Analysis Confirm the analytical method’s accuracy/resoluton. Is it accurate during the exponential phase of PCR?

Table 11.3(Continued) To Enhance this Manipulate One or More of These Components Use appropriate controls. Repeat experiments when data are outside f standard deviation limits Minimize the manipulations from start to Check that the temperature profile is consistent at every position in the heating Control Confirm that controls have similar sequence rofile and amplification efficien Confirm that PCR was linear by producing Analysis Use an automated system to reduce handling steps Detection Check the detection strategys senitivity and ability to measure vield in the al phase of PCR Confirm that the technique has high High-throughput Instrument Select a system that handles microtiter plates and multiple samp multaneously Reaction Use a hot-start PCR strategy(d'aquilla et al., 1991; Chous et al., 1992: Kellogg Use a master PCR reagent mix me aterial: dont different lots. Sample preparation Use of robotics. Storage of sample as cDNA or ethanol precipitate, rather than RNA in solution. Cycling Use one cycling strategy for all samples. Analysis Use an automated system. Detection Use an automated detection system to monitor the exponential phase. Detection Monitor specific PCR product formation by hybridization via nucleic acid probe. Use fluorescent intercalating dye nested PCR t al., 1990). Note: Sensitivity is gained at the expense of quantitation. Use a hot-start PCR strategy Use UNG and dUTp to prevent carryover Analysis Use a real time PCR strategy that detects low levels of amplicon missed by gel PCR 299

PCR 299 Table 11.3 (Continued) To Enhance This Parameter Manipulate One or More of These Components Use appropriate controls. Repeat experiments when data are outside of standard deviation limits. Minimize the manipulations from start to finish. Cycler Check that the temperature profile is consistent at every position in the heating block. Control Confirm that controls have similar sequence profile and amplification efficiency. Confirm that PCR was linear by producing a standard curve. Analysis Use an automated system to reduce handling steps. Detection Check the detection strategy’s senitivity and ability to measure yield in the exponential phase of PCR. Confirm that the technique has high sensitivity and magnitude over a wide dynamic range. High-throughput Instrument Select a system that handles microtiter plates and multiple sample simultaneously. Reaction Use a hot-start PCR strategy (D’Aquilla et al., 1991; Chous et al., 1992; Kellogg et al., 1994). Use a master PCR reagent mix. Use aliquots taken from the same lot of material; don’t mix aliquots from different lots. Sample preparation Use of robotics. Storage of sample as cDNA or ethanol precipitate, rather than RNA in solution. Cycling Use one cycling strategy for all samples. Decrease the cycling time. Analysis Use an automated system. Detection Use an automated detection system to monitor the exponential phase. Sensitivity Detection Monitor specific PCR product formation by hybridization via nucleic acid probe. Use fluorescent intercalating dye (Wittwer et al., 1997). Reaction Use a nested PCR strategy (Simmonds et al., 1990). Note: Sensitivity is gained at the expense of quantitation. Use a hot-start PCR strategy. Use UNG and dUTP to prevent carryover. Analysis Use a real time PCR strategy that detects low levels of amplicon missed by gel

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