Methods in Molecular Biology TM Methods in Molecular Biology TM PCR Protocols S ECOND E DITION Edited by John M. S. Bartlett David Stirling Volume 226 PCR Protocols S ECOND E DITION Edited by John M. S. Bartlett David Stirling i Contents 1. A Short History of the Polymerase Chain Reaction 3 John M. S. Bartlett and David Stirling 2. PCR Patent Issues 7 Peter Carroll and David Casimir 3. Equipping and Establishing a PCR Laboratory 15 Susan McDonagh 4. Quality Control in PCR 20 David Stirling 5. Extraction of Nucleic Acid Templates 27 John M. S. Bartlett 6. Extraction of DNA from Whole Blood 29 John M. S. Bartlett and Anne White 7. DNA Extraction from Tissue 33 Helen Pearson and David Stirling 8. Extraction of DNA from Microdissected Archival Tissues 35 James J. Going 9. RNA Extraction from Blood 43 Helen Pearson 10. RNA Extraction from Frozen Tissue 45 John M. S. Bartlett 11. RNA Extraction from Tissue Sections 47 Helen Pearson 12. Dual DNA/RNA Extraction 49 David Stirling and John M. S. Bartlett 13. DNA Extraction from Fungi, Yeast, and Bacteria 53 David Stirling 14. Isolation of RNA Viruses from Biological Materials 55 Susan McDonagh 15. Extraction of Ancient DNA 57 Wera M. Schmerer 16. DNA Extraction from Plasma and Serum 63 David Stirling 17. Technical Notes for the Detection of Nucleic Acids 65 John M. S. Bartlett 18. Technical Notes for the Recovery and Purificationof PCR Products from Acrylamide Gels 77 David Stirling 19. PCR Primer Design 81 David L. Hyndman and Masato Mitsuhashi 20. Optimization of Polymerase Chain Reactions 89 Haiying Grunenwald 21. Subcycling PCR for Long-Distance Amplificationsof Regions with High and Low Guanine–Cystine Content Amplification of the Intron 22 Inversion of the FVIII Gene David Stirling 101 22. Rapid Amplification of cDNA Ends ii Xin Wang and W. Scott Young III 105 23. Randomly Amplified Polymorphic DNA Fingerprinting The Basics 117 Ranil S. Dassanayake and Lakshman P. Samaranayake 24. Microsphere-Based Single NucleotidePolymorphism Genotyping 123 Marie A. Iannone, J. David Taylor, Jingwen Chen, May-Sung Li,Fei Ye, and Michael P. Weiner 25. Ligase Chain Reaction 135 William H. Benjamin, Jr., Kim R. Smith, and Ken B. Waites 26. Nested RT-PCR in a Single Closed Tube 151 Antonio Olmos, Olga Esteban, Edson Bertolini, and Mariano Cambra 27. Direct PCR from Serum 161 kenji Abe 28. Long PCR Amplification of Large Fragmentsof Viral Genomes 167 A Technical Overview Raymond Tellier, Jens Bukh, Suzanne U. Emerson, and Robert H. Purcell 29. Long PCR Methodology 173 Raymond Tellier, Jens Bukh, Suzanne U. Emerson, and Robert H. Purcell 30. Qualitative and Quantitative PCR A Technical Overview 181 David Stirling 31. Ultrasensitive PCR Detection of Tumor Cells in Myeloma 185 Friedrich W. Cremer and Marion Moos 32. Ultrasensitive Quantitative PCR to Detect RNA Viruses 197 Susan McDonagh 33. Quantitative PCR for cAMP RI Alpha mRNA 205 Use of Site-Directed Mutation and PCR Mimics John M. S. Bartlett 34. Quantitation of Multiple RNA Species 211 Ron Kerr 35. Differential Display A Technical Overview 217 John M. S. Bartlett 36. AU-Differential Display, Reproducibilityof a Differential mRNA Display Targeted to AU Motifs 225 Orlando Dominguez, Lidia Sabater, Yaqoub Ashhab,Eva Belloso, and Ricardo Pujol-Borrell 37. PCR Fluorescence Differential Display 237 Kostya Khalturin, Sergej Kuznetsov, and Thomas C. G. Bosch 38. Microarray Analysis Using RNA Arbitrarily Primed PCR 245 Steven Ringquist, Gaelle Rondeau, Rosa-Ana Risques,Takuya Higashiyama, Yi- Peng Wang, Steffen Porwollik,David Boyle, Michael McClelland, and John Welsh 39. Oligonucleotide Arrays for Genotyping 255 Enzymatic Methods for Typing Single Nucleotide Polymorphisms and Short Tandem Repeats Stephen Case-Green, Clare Pritchard, and Edwin Southern 40. Serial Analysis of Gene Expression 271 Karin A. Oien 41. Mutation and Polymorphism Detection A Technical Overview 287 Joanne Edwards and John M. S. Bartlett 42. Combining Multiplex and Touchdown PCRfor Microsatellite Analysis 295 Kanokporn Rithidech and John J. Dunn iii 43. Detection of Microsatellite Instability and Lossof Heterozygosity Using DNA Extracted fromFormalin-Fixed Paraffin-Embedded Tumor Materialby Fluorescence- Based Multiplex Microsatellite PCR 301 Joanne Edwards and John M. S. Bartlett 44. Reduction of Shadow Band Synthesis DuringPCR Amplification of Repetitive Sequencesfrom Modern and Ancient DNA 309 Wera M. Schmerer 45. Degenerate Oligonucleotide-Primed PCR 315 Michaela Aubele and Jan Smida 46. Mutation Detection Using RT-PCR-RFLP Hitoshi Nakashima, Mitsuteru Akahoshi, and Yosuke Tanaka 319 47. Multiplex Amplification RefractoryMutation System for the Detectionof Prothrombotic Polymorphisms 323 David Stirling 48. PCR-SSCP Analysis of Polymorphism 328 A Simple and Sensitive Method for Detecting DifferencesBetween Short Segments of DNA Mei Han and Mary Ann Robinson 49. Sequencing A Technical Overview 337 David Stirling 50. Preparation and Direct Automated Cycle Sequencingof PCR Products 341 Susan E. Daniels 51. Nonradioactive PCR Sequencing Using Digoxigenin 347 Siegfried Kösel, Christoph B. Lücking, Rupert Egensperger,and Manuel B. Graeber 52. Direct Sequencing by Thermal Asymmetric PCR 355 Georges-Raoul Mazars and Charles Theillet 53. Analysis of Nucleotide Sequence Variationsby Solid-Phase Minisequencing 361 Anu Suomalainen and Ann-Christine Syvänen 54. Direct Sequencing with Highly Degenerateand Inosine-Containing Primers 367 Zhiyuan Shen, Jingmei Liu, Robert L. Wells, and Mortimer M. Elkind 55. Determination of Unknown Genomic SequencesWithout Cloning 373 Jean-Pierre Quivy and Peter B. Becker 56. Cloning PCR Products for Sequencing in M13 Vectors 385 David Walsh 57. DNA Rescue by the Vectorette Method 393 Marcia A. McAleer, Alison J. Coffey, and Ian Dunham 58. Technical Notes for Sequencing Difficult Templates 401 David Stirling 59. PCR-Based Detection of Nucleic Acidsin Chromosomes, Cells, and Tissues 405 Technical Considerations on PRINS and In Situ PCR and Comparison with In Situ Hybridization Ernst J. M. Speel, Frans C. S. Ramaekers, and Anton H. N. Hopman 60. Cycling Primed In Situ Amplification 425 John H. Bull and Lynn Paskins 61. Direct and Indirect In Situ PCR 433 Klaus Hermann Wiedorn and Torsten Goldmann 62. Reverse Transcriptase In Situ PCR New Methods in Cellular Interrogation 445 Mark Gilchrist and A. Dean Befus iv 63. Primed In Situ Nucleic Acid Labeling Combined with Immunocytochemistry to Simultaneously Localize DNA and Proteins in Cells and Chromosomes 453 Ernst J. M. Speel, Frans C. S. Ramaekers, and Anton H. N. Hopman 64. Cloning and Mutagenesis A Technical Overview 467 Helen Pearson and David Stirling 65. Using T4 DNA Polymeraseto Generate Clonable PCR Products 469 Kai Wang 66. A T-Linker Strategy for Modificationand Directional Cloning of PCR Products 475 Robert M. Horton, Raghavanpillai Raju, and Bianca M. Conti-Fine 67. Cloning Gene Family Members Using PCRwith Degenerate Oligonucleotide Primers Gregory M. Preston 485 68. cDNA Libraries from a Low Amount of Cells 499 Philippe Ravassard, Christine Icard-Liepkalns, Jacques Mallet,and Jean Baptiste Dumas Milne Edwards 69. Creation of Chimeric Junctions, Deletions, and Insertions by PCR 511 Genevieve Pont-Kingdon 70. Recombination and Site-Directed MutagenesisUsing Recombination PCR 517 Douglas H. Jones and Stanley C. Winistorfer 71. Megaprimer PCR Application in Mutagenesis and Gene Fusion 525 Emily Burke and Sailen Barik 3 From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ 1 A Short History of the Polymerase Chain Reaction John M. S. Bartlett and David Stirling The development of the polymerase chain reaction (PCR) has often been likened to the development of the Internet, and although this does risk overstating the impact of PCR outside the scientic community, the comparison works well on a number of levels. Both inventions have emerged in the last 20 years to the point where it is difcult to imagine life without them. Both have grown far beyond the connes of their original simple design and have created opportunities unimaginable before their invention. Both have also spawned a whole new vocabulary and professionals literate in that vocabulary. It is hard to believe that the technique that formed the cornerstone of the human genome project and is fundamental to many molecular biology laboratory protocols was discovered only 20 years ago. For many, the history and some of the enduring controversies are unknown yet, as with the discovery of the structure of DNA in the 1950s, the discovery of PCR is the subject of claim and counterclaim that has yet to be fully resolved. The key stages are reviewed here in brief for those for whom both the history and application of science holds interest. The origins of PCR as we know it today sprang from key research performed in the early 1980s at Cetus Corporation in California. The story is that in the spring of 1983, Kary Mullis had the original idea for PCR while cruising in a Honda Civic on Highway 128 from San Francisco to Mendocino. This idea claimed to be the origin of the modern PCR technique used around the world today that forms the foundation of the key PCR patents. The results for Mullis were no less satisfying; after an initial $10,000 bonus from Cetus Corporation, he was awarded the 1993 Nobel Prize for chemistry. The original concept for PCR, like many good ideas, was an amalgamation of several components that were already in existence: The synthesis of short lengths of single-stranded DNA (oligonucleotides) and the use of these to direct the target-specic synthesis of new DNA copies using DNA polymerases were already standard tools in the repertoire of the molecular biologists of the time. The novelty in Mullis’s concept was using the juxtaposition of two oligonucleotides, complementary to opposite strands of the DNA, to specically amplify the region between them and to achieve this in a repetitive manner so that the product of one round of polymerase activity was added to the pool of template for the next round, hence the chain reaction. In his History of PCR (1), Paul Rabinow quotes Mullis as saying: History of PCR 3 The thing that was the “Aha!” the “Eureka!” thing about PCR wasn’t just putting those [things] together…the remarkable part is that you will pull out a little piece of DNA from its context, and that’s what you will get amplied. That was the thing that said, “you could use this to isolate a fragment of DNA from a complex piece of DNA, from its context.” That was what I think of as the genius thing.…In a sense, I put together elements that were already there.…You can’t make up new elements, usually. The new element, if any, it was the combination, the way they were used.…The fact that I would do it over and over again, and the fact that I would do it in just the way I did, that made it an invention…the legal wording is “presents an unanticipated solution to a long-standing problem,” that’s an invention and that was clearly PCR. In fact, although Mullis is widely credited with the original invention of PCR, the successful application of PCR as we know it today required considerable further development by his colleagues at Cetus Corp, including colleagues in Henry Erlich’s lab (2–4), and the timely isolation of a thermostable polymerase enzyme from a thermophilic bacterium isolated from thermal springs. Furthermore, challenges to the PCR patents held by Hoffman La Roche have claimed at least one incidence of “prior art,” that is, that the original invention of PCR was known before Mullis’s work in the mid-1980s. This challenge is based on early studies by Khorana et al. in the late 1960s and early 1970s (see chapter 2). Khorana’s work used a method that he termed repair replication, and its similarity to PCR can be seen in the following steps: (1) annealing of primers to templates and template extension; (2) separation of the newly synthesized strand from the template; and (3) re-annealing of the primer and repetition of the cycle. Readers are referred to an extensive web-based literature on the patent challenges arising from this “prior art” and to chapter 2 herein for further details. Whatever the nal outcome, it is clear that much of the work that has made PCR such a widely used methodology arose from the laboratories of Mullis and Erlich at Cetus in the mid-1980s. The DNA polymerase originally used for the PCR was extracted from the bacterium Escherichia coli. Although this enzyme had been a valuable tool for a wide range of applications and had allowed the explosion in DNA sequencing technologies in the preceding decade, it had distinct disadvantages in PCR. For PCR, the reaction must be heated to denature the double-stranded DNA product after each round of synthesis. Unfortunately, heating also irreversibly inactivated the E. coli DNA polymerase, and therefore fresh aliquots of enzyme had to be added by hand at the start of each cycle. What was required was a DNA polymerase that remained stable during the DNA denaturation step performed at around 95°C. The solution was found when the bacterium Thermophilus aquaticus was isolated from hot springs, where it survived and proliferated at extremely high temperatures, and yielded a DNA polymerase that was not rapidly inactivated at high temperatures. Gelfand and his associates at Cetus puried and subsequently cloned this polymerase (5,6), allowing a complete PCR amplication to be created without opening the reaction tube. Furthermore, because the enzyme was isolated from a thermophilic organism, it functioned optimally at tem- perature of around 72°C, allowing the DNA synthesis step to be performed at higher temperatures than was possible with the E. coli enzyme, which ensured that the template DNA strand could be copied with higher delity as the result of a greater stringency of primer binding, eliminating the nonspecic products that had plagued earlier attempts at PCR amplication. 4 Bartlett and Stirling However, even with this improvement, the PCR technique was laborious and slow, requiring manual transfer between water baths at different temperatures. The rst thermocycling machine, “Mr Cycle,” which replicated the temperature changes required for the PCR reaction without the need for manual transfer, was developed by Cetus to facilitate the addition of fresh thermolabile polymerases. After the purication of Taq polymerase, Cetus and Perkin–Elmer introduced the closed DNA thermal cyclers that are widely used today (7). That PCR has become one of the most widely used tools in molecular biology is clear from Fig. 1. What is not clear from this simplistic analysis of the literature is the huge range of questions that PCR is being used to answer. Another scientist at Cetus, Stephen Scharf, is quoted as stating that …the truly astonishing thing about PCR is precisely that it wasn’t designed to solve a problem; once it existed, problems began to emerge to which it could be applied. One of PCR’s distinctive characteristics is unquestionably its extraordinary versatility. That versatility is more than its ‘applicability’ to many different situations. PCR is a tool that has the power to create new situations for its use and those required to use it. More than 3% of all PubMed citations now refer to PCR (Fig. 2). Techniques have been developed in areas as diverse as criminal forensic investigations, food science, ecological eld studies, and diagnostic medicine. Just as diverse are the range of adaptations and variations on the original theme, some of which are exemplied in this volume. The enormous advances made in our understanding of the human genome (and that of many other species), would not have been possible, where it not for the remarkable simple and yet exquisitely adaptable technique which is PCR. Fig. 1. Results of a PubMed search for articles containing the phrase “Polymerase Chain Reaction.” Graph shows number of articles listed in each year. History of PCR 5 References 1. Rabinow, P. (1996) Making PCR: A Story of Biotechnology. University of Chicago Press, Chicago. 2. Saiki, R., Scharf, S., Faloona, F., Mullis, K., Horn, G., and Erlich, H. (1985) Enzymatic amplication of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–1354. 3. Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G., and Erlich, H. (1986) Specic enzymatic amplication of DNA in vitro: The polymerase chain reaction. Cold Spring Harbor Symp. Quant. Biol. 51, 263–273. 4. Mullis, K. and Faloona, F. (1987) Specic synthesis of DNA in vitro via a polymerase- catalyzed chain reaction. Methods Enzymol. 155, 335–350. 5. Saiki, R., Gelfand, D., Stoffel, S., Scharf, S., Higuchi, R., Horn, et al. (1988) Primer- directed enzymatic amplication of DNA with a thermostable DNA polymerase. Science 239, 487–491. 6. Lawyer, F., Stoffer, S, Saiki, R., Chang, S., Landre, P., Abramson, R., et al. (1993) High- level expression, purication, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form decient in 5′ to 3′ exonuclease activity. PCR Methods Appl. 2, 275–287. 7. http://www.si.edu/archives/ihd/videocatalog/9577.htm Fig. 2. Results of a PubMed search for articles containing the phrase “Polymerase Chain Reaction.” Graph shows number of articles listed in each year expressed as a percentage of the total PubMed citations for each year. 6 Bartlett and Stirling 7 From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ 2 PCR Patent Issues Peter Carroll and David Casimir 1. Introduction The science of the so-called polymerase chain reaction (PCR) is now well known. However, the legal story associated with PCR is, for the most part, not understood and constantly changing. This presents difculties for scientists, whether in academia or industry, who wish to practice the PCR process. This chapter summarizes the major issues related to obtaining rights to practice PCR. The complexity of the patent system is explained with a few PCR-specic examples highlighted. The chapter also provides an overview of the exemption or exception from patent infringement associated with certain bona-de researchers and discusses the status of certain high-prole patents covering aspects of the PCR process. 2. Intellectual Property Rights Various aspects of the PCR process, including the method itself, are protected by patents in the United States and around the world. As a general rule, patents give the patent owner the exclusive right to make, use, and sell the compositions or process claimed by the patent. If someone makes, uses, or sells the patented invention in a country with an issued patent, the patent owner can invoke the legal system of that country to stop future infringing activities and possibly recover money from the infringer. A patent owner has the right to allow, disallow, or set the terms under which people make, use, and sell the invention claimed in their patents. In an extreme situation, a patent owner can exclude everyone from making, using, and selling the invention, even under conditions where the patent owner does not produce the product themselves—effectively removing the invention from the public for the lifetime of the patent (typically 20 years from the ling date of the patent). If a patent owner chooses to allow others to make, use, or sell the invention, they can contractually control nearly every aspect of how the invention is disbursed to the public or to certain companies or individuals, so long as they are not unfairly controlling products not covered by the patent. For example, a patent owner can select or exclude certain elds of use for methods like PCR (e.g., research use, clinical use, etc.) while allowing others. There are an extraordinary number of patents related to the PCR technology. For example, in the United States alone, there are more than 600 patents claiming aspects PCR Patent Issues 7 [...]... should be assembled and placed on the appropriate thermocycler After cycling, these are removed to the secondary PCR area, where reactions are assembled and placed on cyclers dedicated for this process Other automated/integrated/single-round equipment should be positioned with secondary thermocyclers to reduce the risk of contamination (see Note 6) 3.4 Post -PCR Processing All final amplified products should... earliest and most basic PCR- related patents A full analysis of the hundreds of PCR- related patents is not practical in an article this size, let alone a multivolume treatise It is hoped that the following discussion will provide a preliminary framework for understanding the broad PCR patent landscape The early PCR patents now owned by Hoffmann-LaRoche have been aggressively enforced In particular, the... reflected in the cost For example, if basic PCR is all that is required, equipment from the lower end of the range might suffice These machines have programmable From: Methods in Molecular Biology, Vol 226: PCR Protocols, Second Edition Edited by: J M S Bartlett and D Stirling © Humana Press Inc., Totowa, NJ 15 16 McDonagh Fig 1 Unidirectional flow in a PCR laboratory blocks, often with a heated lid,... provide detailed reviews of methods for nucleic acid extraction and some recommended web sites are listed in the reference section From: Methods in Molecular Biology, Vol 226: PCR Protocols, Second Edition Edited by: J M S Bartlett and D Stirling © Humana Press Inc., Totowa, NJ 27 28 Bartlett References 1 US Department of Commerce Molecular Biology Techniques Forum http://research nwfsc.noaa.gov /protocols/ methods/... variability? The design of optimum PCR primers will be discussed later It is important to remember, however, that these are single-stranded DNA molecules and are therefore relatively labile Repeated freeze/thawing will cause degradation to shorter products, From: Methods in Molecular Biology, Vol 226: PCR Protocols, Second Edition Edited by: J M S Bartlett and D Stirling © Humana Press Inc., Totowa, NJ 21...8 Carroll and Casimir of PCR Such patents cover the basic methods itself (originally owned by Cetus Corporation and now owned by Hoffmann-LaRoche), thermostable polymerases useful in PCR, as well as many non -PCR applications, (e.g., Taq polymerase, Tth polymerase, Pfu polymerase, KOD polymerase, Tne polymerase, Tma polymerase, modified polymerases, etc.), devices used in PCR (e.g., thermocyclers,... heated to seal the end and curled to form a “loop” or “hook” for spooling DNA EDTA (0.5 M), pH 8.0: Add 146.1 g of anhydrous EDTA to 800 mL of distilled water Adjust pH to 8.0 with NaOH pellets (this will require about 20 g) Make up to 1 L with distilled water Autoclave at 15 p.s.i for 15 min 1 M Tris-HCl, pH 7.6: Dissolve 121.1 g of Tris base in 800 mL of distilled water Adjust pH with concentrated... Tris-HCl, 150 mM NaCl, 0.06 M EDTA, 1% sodium dodecyl sulphate, pH 8.0 Take 400 mL of 1 M Tris (pH 7.6), 120 mL of 0.5 M EDTA (pH 8.0), 8.76 g of NaCl, and adjust pH to 8.0 Make up to 1 L with distilled water Autoclave 15 min at 15 p.s.i After autoclaving, add 10 g of sodium dodecyl sulphate From: Methods in Molecular Biology, Vol 226: PCR Protocols, Second Edition Edited by: J M S Bartlett and D Stirling... respect to these patents are most relevant and are focused on herein 3.1 Obtaining Rights to Practice PCR In the case of the early PCR patents, Hoffmann-LaRoche, directly and through certain designated partners, has made PCR available to the public under specific conditions, depending on the intended use of the method (see for availability of licenses and current... tips can be used for post -PCR steps 3 An ultraviolet irradiation source is valuable in reducing contamination; however, Cimino et al (10) recommend caution when using this method alone Otherwise, wash down all nonmetal surfaces with 0.1 N HCl, or 10% bleach, followed by water 4 Nitrile gloves should be used for safety when handling ethidium bromide if used in gel electrophoresis 5 As kit-based formats . polymerase, Tth polymerase, Pfu polymerase, KOD polymerase, Tne polymerase, Tma polymerase, modi ed polymerases, etc.), devices used in PCR (e.g., thermocyclers, sample tubes and vessels, solid supports,. Navigating the PCR Patent Mineeld The following discussion focuses on issues related to the earliest and most basic PCR- related patents. A full analysis of the hundreds of PCR- related patents is not. Microsatellite Instability and Lossof Heterozygosity Using DNA Extracted fromFormalin-Fixed Paraffin-Embedded Tumor Materialby Fluorescence- Based Multiplex Microsatellite PCR 301 Joanne Edwards