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 AoyagiHow 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