PCR Protocols

519 Pages·2003·14.425 MB·English

by  John M. S. Bartlett, David Stirling (auth.), John M. S. Bartlett, David Stirling (eds.)

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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
i
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
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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
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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
iv
History of PCR 3
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 scientific 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
difficult to imagine life without them. Both have grown far beyond the confines 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-specific
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 specifically 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:
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
3
4 Bartlett and Stirling
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 amplified. 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
final 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
purified and subsequently cloned this polymerase (5,6), allowing a complete PCR
amplification 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 fidelity as the result of a greater
stringency of primer binding, eliminating the nonspecific products that had plagued
earlier attempts at PCR amplification.
History of PCR 5
Fig. 1. Results of a PubMed search for articles containing the phrase “Polymerase Chain
Reaction.” Graph shows number of articles listed in each year.
However, even with this improvement, the PCR technique was laborious and slow,
requiring manual transfer between water baths at different temperatures. The first
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 purification 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 field studies, and diagnostic medicine. Just as diverse are the range of
adaptations and variations on the original theme, some of which are exemplified 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.
6 Bartlett and Stirling
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.
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
amplification 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) Specific
enzymatic amplification of DNA in vitro: The polymerase chain reaction. Cold Spring
Harbor Symp. Quant. Biol. 51, 263–273.
4. Mullis, K. and Faloona, F. (1987) Specific 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 amplification 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, purification, and enzymatic characterization of full-length Thermus
aquaticus DNA polymerase and a truncated form deficient in 5′ to 3′ exonuclease activity.
PCR Methods Appl. 2, 275–287.
7. http://www.si.edu/archives/ihd/videocatalog/9577.htm
PCR Patent Issues 7
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 difficulties 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-specific examples highlighted. The chapter also provides
an overview of the exemption or exception from patent infringement associated with
certain bona-fide researchers and discusses the status of certain high-profile 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 filing 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 fields 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
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
7