Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S Gerstein opyright◎2001 ISBNS:0-471-37972-7( Paper);0-471 (Electronic) PCR Kazuko Aoyag Introduction 292 Developing a PCR Strategy: The Project Stage 293 Assess Your needs Identify Any Weak Links in Your PCR Strategy 295 Manipulate the Reaction to Meet Your Needs Developing a PCr Strategy: The Experimental Stage 296 What Are the Practical Criteria for Evaluating a DNA Polymerase for Use in PCR! 296 How Can Nucleotides and Primers Affect a PCr Reaction How Do the Components of a Typical PCR Reaction Buffer Affect the reaction? How Can You Minimize the Frequency of Template What makes for good pos and Negative Amplification Controls? What makes for a reliable control for gene Expression? 309 ifferent Cycling Parameters Af PCR Reaction? Instrumentation: By What Criteria Could You Evaluate a Thermocycler? 309 How Can Sample Preparation Affect Your results?
11 PCR Kazuko Aoyagi Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 Developing a PCR Strategy: The Project Stage . . . . . . . . . . . . . 293 Assess Your Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293 Identify Any Weak Links in Your PCR Strategy . . . . . . . . . . 295 Manipulate the Reaction to Meet Your Needs . . . . . . . . . . 296 Developing a PCR Strategy: The Experimental Stage . . . . . . . 296 What Are the Practical Criteria for Evaluating a DNA Polymerase for Use in PCR? . . . . . . . . . . . . . . . . . . . . . . . . 296 How Can Nucleotides and Primers Affect a PCR Reaction? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303 How Do the Components of a Typical PCR Reaction Buffer Affect the Reaction? . . . . . . . . . . . . . . . . . . . . . . . . . 305 How Can You Minimize the Frequency of Template Contamination? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306 What Makes for Good Positive and Negative Amplification Controls? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308 What Makes for A Reliable Control for Gene Expression? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 How Do the Different Cycling Parameters Affect a PCR Reaction? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 Instrumentation: By What Criteria Could You Evaluate a Thermocycler? . . . . . . . . . . . . . . . . . . . . . . . . . . 309 How Can Sample Preparation Affect Your Results? . . . . . . . 311 291 Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S. Gerstein Copyright © 2001 by Wiley-Liss, Inc. ISBNs: 0-471-37972-7 (Paper); 0-471-22390-5 (Electronic)
How Can You distinguish between an Inhibitor Carried over with the Template and Modification of he dNA Template 32 What Are the Steps to Good Primer Design Which Detection and Analysis Strategy Best Meets Your needs? Troubleshooting∵ 315 RT-PCR Summ 322 Bibliography Appendix A: Preparation of Plasmid DNA for Use as PCR ontrols in Multiple Experiments Appendix B: Computer Software for Selecting Primers Appendix C: BLAST Searches 328 AppendⅸD: Useful Web sites∴ 28 NTRODUCTION The principle of the polymerase chain reaction(PCR) was first reported in 1971(Kleppe et al, 1971), but it was only after the dis- covery of the thermostable Taq dna polymerase (Saiki et al., 1988: Lawyer et al., 1989) that this technology became easy to use Initially the thermal cycling was handled manually by transferring samples to be amplified from one water bath to another with the addition of fresh enzyme per cycle after the denaturation step (Saiki et aL., 1986: Mullis et al., 1986). Today, 30 years later, we are fortunate to have thermal cyclers, along with enzymes and other reagents dedicated for various PCR applications. The advances in PCR technology and the number of annual publica- tions using PCR in some area of the research has grown tremen dously from a single-digit number to 1.6x 10 in 1999(Medline search). The popularity of the PCr method lies in its simplicity, which permits even a lay person without a molecular biology degree to run a reaction with minimum training However, this easy"“ entry” can also act as a"trap” to encounter ommon problems with this technology. The purpose of this chapter is to help you select and optimize the most appropriate PCR strategy, to avoid problems, and to help you think your way out of problems that do arise. While your research topic may be unique, the solutions to most PCR problems are less so. Employ ing one or a combination of methods mentioned in this chapter could solve problems. I encourage readers to spend time in setting up the lab, choosing the appropriate protocol, optimizing the con 292 Aoyagi
How Can You Distinguish between an Inhibitor Carried over with the Template and Modification of the DNA Template? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312 What Are the Steps to Good Primer Design? . . . . . . . . . . 312 Which Detection and Analysis Strategy Best Meets Your Needs? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315 RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322 Appendix A: Preparation of Plasmid DNA for Use as PCR Controls in Multiple Experiments . . . . . . . . . . . . . . . . . . . . . . . . 327 Appendix B: Computer Software for Selecting Primers . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . 327 Appendix C: BLAST Searches . . . . . . . . . . . . . . . . . . . . . . . . . . . 328 Appendix D: Useful Web Sites . . . . . . . . . . . . . . . . . . . . . . . . . 328 INTRODUCTION The principle of the polymerase chain reaction (PCR) was first reported in 1971 (Kleppe et al., 1971), but it was only after the discovery of the thermostable Taq DNA polymerase (Saiki et al., 1988; Lawyer et al., 1989) that this technology became easy to use. Initially the thermal cycling was handled manually by transferring samples to be amplified from one water bath to another with the addition of fresh enzyme per cycle after the denaturation step (Saiki et al., 1986; Mullis et al., 1986). Today, 30 years later, we are fortunate to have thermal cyclers, along with enzymes and other reagents dedicated for various PCR applications. The advances in PCR technology and the number of annual publications using PCR in some area of the research has grown tremendously from a single-digit number to 1.6 ¥ 104 in 1999 (Medline search). The popularity of the PCR method lies in its simplicity, which permits even a lay person without a molecular biology degree to run a reaction with minimum training. However, this easy “entry” can also act as a “trap” to encounter common problems with this technology. The purpose of this chapter is to help you select and optimize the most appropriate PCR strategy, to avoid problems, and to help you think your way out of problems that do arise. While your research topic may be unique, the solutions to most PCR problems are less so. Employing one or a combination of methods mentioned in this chapter could solve problems. I encourage readers to spend time in setting up the lab, choosing the appropriate protocol, optimizing the con- 292 Aoyagi
ditions and analysis method before running the first PCr reaction In the long run, you will save time and resources. This chapter provides practical guidelines and references to in- depth information. Other useful information is added in the Appendix to help you navigate through various tools available in today's market DEVELOPING A PCR STRATEGY: THE PROJECT STAGE Assess Your needs First ask yourself what outcome you need to achieve to feel suc cessful with your experiment(Table 11.1). What kind of informa tion do you need to get? Is it qualitative or quantitative? Are you tting up a routine analysis to run for the next two years, or is this for the manuscript you need to send to the editor in a hurry in order for your paper to get accepted? Your priorities will help you choose the method that best fits your needs. Table 11.2a shows an example of a list for a researcher who needs to develop a PCr method where approximately 48 genes will be studied for relative gene expression in response to various drug treatments to be given over a three-year period. In contrast Table 11.2b shows a list of a scientist who wishes to clone a gene with two different mRNA forms generated by alternative splicing Table II. Priority Check List High/Medium/Low Quantitative Sensitivity Fidelity High-throughput Reproducibility Cost-sensitive Long PCR product Limited available starting material Short template size Gel based Simple method tivity iny Automated DNA PCR RNA PCR Itiple samples Mul PCR 293
ditions and analysis method before running the first PCR reaction. In the long run, you will save time and resources. This chapter provides practical guidelines and references to indepth information. Other useful information is added in the Appendix to help you navigate through various tools available in today’s market. DEVELOPING A PCR STRATEGY:THE PROJECT STAGE Assess Your Needs First ask yourself what outcome you need to achieve to feel successful with your experiment (Table 11.1). What kind of information do you need to get? Is it qualitative or quantitative? Are you setting up a routine analysis to run for the next two years, or is this for the manuscript you need to send to the editor in a hurry in order for your paper to get accepted? Your priorities will help you choose the method that best fits your needs. Table 11.2a shows an example of a list for a researcher who needs to develop a PCR method where approximately 48 genes will be studied for relative gene expression in response to various drug treatments to be given over a three-year period. In contrast, Table 11.2b shows a list of a scientist who wishes to clone a gene with two different mRNA forms generated by alternative splicing PCR 293 Table 11.1 Priority Check List Objectives High/Medium/Low Quantitative Sensitivity Fidelity High-throughput Reproducibility Cost-sensitive Long PCR product Limited available starting material Short template size Gel based Simple method Nonradioactivity involved Automated Long-term project DNA PCR RNA PCR Multiple samples Multiplex
Table 1 1.2a Priority List: Researcher I Objecti High/Medium/Low H ensitivity High-throughput Long PCR product Limited available hort template size method dioactivity involved ong-term project A PCR NA PCR LMHLHHHHLHHH ultiple Table I 1. 2b Priority List: Researcher 2 Obiective High/Medium/Low Sensitivity High-throughput Reproducibility HLHH Cost-sensitive Long pcr product Limited available Short template size el based Simple method Nonradioactivity involved Automated Long-term project DNA PCR RNA PCR HLLLLLHLL Multiple samples Multiplex mechanisms. The purpose of Researcher 2 is to amplify the cDNA and to demonstrate the size difference by separating the two forms by gelelectrophoresis. The data are needed for a manuscript due in two months. You can see the differences between the pri- orities and needs of the two researchers Aoyagi
mechanisms. The purpose of Researcher 2 is to amplify the cDNA and to demonstrate the size difference by separating the two forms by gel electrophoresis.The data are needed for a manuscript due in two months. You can see the differences between the priorities and needs of the two researchers. 294 Aoyagi Table 11.2a Priority List: Researcher 1 Objectives High/Medium/Low Quantitative H Sensitivity H Fidelity M High-throughput M Reproducibility H Cost-sensitive M Long PCR product L Limited available M starting material Short template size H Gel based L Simple method H Nonradioactivity involved H Automated H Long-term project H DNA PCR L RNA PCR H Multiple samples H Multiplex H Table 11.2b Priority List: Researcher 2 Objectives High/Medium/Low Quantitative M Sensitivity M Fidelity H High-throughput L Reproducibility H Cost-sensitive H Long PCR product L Limited available M starting material Short template size M Gel based H Simple method L Nonradioactivity involved L Automated L Long-term project L DNA PCR L RNA PCR H Multiple samples L Multiplex L
After setting clear objectives of what your PCR reaction must accomplish, check that you have the adequate resources. This includes not only budget but also head count, skill level, time equipment, sequence information, sample supply, and other issues If time is most critical, then you may require a colleague's assis- tance or a new instrument to do the project as quickly as possible In a similar token, if the sample is difficult to obtain in abundance the choice of PCr that minimizes the sample requirement becomes more important Selecting one PCR strategy that optimally satisfies every research need is unlikely. At this early planning stage, a compro- mise between competing needs will likely be required. Remem ber that after all the planning is complete, the final PCR strategy still has to evolve at the lab bench Identify Any Weak Links in Your PCR Strategy A There are many parameters that affect the outcome of a PCR action some examples are as follows: PCR reaction chemistry(enzyme, nucleotide, sample, primer, buffer, additives) PCR instrument type(ramp time, well-to-well homogeneity, capacity to handle many samples) Thermal cycling conditions (two-step three-step, cycle segment length-1. e, denaturation, annealing, and exten sion--ramp time, etc. Sample collection, preparation, and storage (DNA, RNa microdissected tissue, cells, and archived material) PCR primer design Detection method (simultaneous detection, post PCR detection). Analysis method(statistical analysis) Like the weakest link in a chain, your final result will be limited by the parameter that is least optimum. For example, suppose that you're studying the tissue-specific regulation of two mRNA forms Regardless of the time spent optimizing the PCr reaction, instru- ment type, and everything to near-perfection, the use of agarose gel electrophoresis may not allow you to reach the conclusion that there are two different mRNA forms if their molecular weight are similar. You might require a separation technique with greater resolving power Suppose that your objective requires quantitative PCR. RNA from 30 samples is collected and RT-PCR is performed. The PCR reaction is run in duplicate and repeated twice on two different PCR
After setting clear objectives of what your PCR reaction must accomplish, check that you have the adequate resources. This includes not only budget but also head count, skill level, time, equipment, sequence information, sample supply, and other issues. If time is most critical, then you may require a colleague’s assistance or a new instrument to do the project as quickly as possible. In a similar token, if the sample is difficult to obtain in abundance, the choice of PCR that minimizes the sample requirement becomes more important. Selecting one PCR strategy that optimally satisfies every research need is unlikely. At this early planning stage, a compromise between competing needs will likely be required. Remember that after all the planning is complete, the final PCR strategy still has to evolve at the lab bench. Identify Any Weak Links in Your PCR Strategy There are many parameters that affect the outcome of a PCR reaction. Some examples are as follows: • PCR reaction chemistry (enzyme, nucleotide, sample, primer, buffer, additives). • PCR instrument type (ramp time, well-to-well homogeneity, capacity to handle many samples). • Thermal cycling conditions (two-step, three-step, cycle segment length—i.e., denaturation, annealing, and extension—ramp time, etc.). • Sample collection, preparation, and storage (DNA, RNA, microdissected tissue, cells, and archived material). • PCR primer design. • Detection method (simultaneous detection, post PCR detection). • Analysis method (statistical analysis). Like the weakest link in a chain, your final result will be limited by the parameter that is least optimum. For example, suppose that you’re studying the tissue-specific regulation of two mRNA forms. Regardless of the time spent optimizing the PCR reaction, instrument type, and everything to near-perfection, the use of agarose gel electrophoresis may not allow you to reach the conclusion that there are two different mRNA forms if their molecular weights are similar.You might require a separation technique with greater resolving power. Suppose that your objective requires quantitative PCR. RNA from 30 samples is collected and RT-PCR is performed. The PCR reaction is run in duplicate and repeated twice on two different PCR 295
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 electrophoresis (PAGE). For some unknown reason, the second experiment shows different quantitative data. Which data are correct? Without a sufficient number of samples to calculate standard 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 synthesis 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 thermostable DNA polymerase. Were you planning to identify a rare mutation in a heterogeneous population as in allelic polymorphisms (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 primerdimer 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 biochemical 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 appropriate enzyme for PCR; experimentation might still be required to determine which enzyme works best. Abu Al-Soud and Radstrom (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 predicting 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