武汉大学生命科学学院：《分子生物学》（英文版）11 PCR

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 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 1.6 ¥ 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. 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

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 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 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 pri￾orities 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 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 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 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 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