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PCR Protocols, Second Edition is a part of Methods in Molecular Biology, Vol. 226, consist of 71 authors, which are: 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) 22. Rapid Amplification of cDNA Ends (Xin Wang and W. Scott Young III) 23. Randomly Amplified Polymorphic DNA Fingerprinting--The Basics (Ranil S. Dassanayake and Lakshman P. Samaranayake) 24. Microsphere-Based Single NucleotidePolymorphism Genotyping (Marie A. Iannone, J. David Taylor, Jingwen Chen, May-Sung Li,Fei Ye, and Michael P. Weiner) 25. Ligase Chain Reaction (William H. Benjamin, Jr., Kim R. Smith, and Ken B. Waites) 26. Nested RT-PCR in a Single Closed Tube (Antonio Olmos, Olga Esteban, Edson Bertolini, and Mariano Cambra) 27. Direct PCR from Serum (kenji Abe) 28. Long PCR Amplification of Large Fragmentsof Viral Genomes ---A Technical Overview (Raymond Tellier, Jens Bukh, Suzanne U. Emerson, and Robert H. Purcell) 29. Long PCR Methodology (Raymond Tellier, Jens Bukh, Suzanne U. Emerson, and Robert H. Purcell) 30. Qualitative and Quantitative PCR-----A Technical Overview (David Stirling) 31. Ultrasensitive PCR Detection of Tumor Cells in Myeloma (Friedrich W. Cremer and Marion Moos) 32. Ultrasensitive Quantitative PCR to Detect RNA Viruses (Susan McDonagh) 33. Quantitative PCR for cAMP RI Alpha mRNA -Use of Site-Directed Mutation and PCR Mimics (John M. S. Bartlett) 34. Quantitation of Multiple RNA Species (Ron Kerr) 35. Differential Display----- A Technical Overview (John M. S. Bartlett) 36. AU-Differential Display, Reproducibilityof a Differential mRNA Display Targeted to AU Motifs (Orlando Dominguez, Lidia Sabater, Yaqoub Ashhab,Eva Belloso, and Ricardo Pujol-Borrell) 37. PCR Fluorescence Differential Display (Kostya Khalturin, Sergej Kuznetsov, and Thomas C. G. Bosch) 38. Microarray Analysis Using RNA Arbitrarily Primed PCR (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 - 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 (Karin A. Oien) 41. Mutation and Polymorphism Detection- A Technical Overview (Joanne Edwards and John M. S. Bartlett) 42. Combining Multiplex and Touchdown PCRfor Microsatellite Analysis (Kanokporn Rithidech and John J. Dunn) 43. Detection of Microsatellite Instability and Lossof Heterozygosity Using DNA Extracted fromFormalin-Fixed Paraffin-Embedded Tumor Materialby Fluorescence- Based Multiplex Microsatellite PCR (Joanne Edwards and John M. S. Bartlett) 44. Reduction of Shadow Band Synthesis DuringPCR Amplification of Repetitive Sequencesfrom Modern and Ancient DNA (Wera M. Schmerer) 45. Degenerate Oligonucleotide-Primed PCR (Michaela Aubele and Jan Smida) 46. Mutation Detection Using RT-PCR-RFLP (Hitoshi Nakashima, Mitsuteru Akahoshi, and Yosuke Tanaka) 47. Multiplex Amplification RefractoryMutation System for the Detectionof Prothrombotic Polymorphisms (David Stirling) 48. PCR-SSCP Analysis of Polymorphism - A Simple and Sensitive Method for Detecting DifferencesBetween Short Segments of DNA (Mei Han and Mary Ann Robinson) 49. Sequencing---- A Technical Overview (David Stirling) 50. Preparation and Direct Automated Cycle Sequencingof PCR Products (Susan E. Daniels ) 51. Nonradioactive PCR Sequencing Using Digoxigenin (Siegfried Kösel, Christoph B. Lücking, Rupert Egensperger,and Manuel B. Graeber) 52. Direct Sequencing by Thermal Asymmetric PCR (Georges-Raoul Mazars and Charles Theillet) 53. Analysis of Nucleotide Sequence Variationsby Solid-Phase Minisequencing (Anu Suomalainen and Ann-Christine Syvänen) 54. Direct Sequencing with Highly Degenerateand Inosine-Containing Primers (Zhiyuan Shen, Jingmei Liu, Robert L. Wells, and Mortimer M. Elkind) 55. Determination of Unknown Genomic SequencesWithout Cloning (Jean-Pierre Quivy and Peter B. Becker) 56. Cloning PCR Products for Sequencing in M13 Vectors (David Walsh) 57. DNA Rescue by the Vectorette Method (Marcia A. McAleer, Alison J. Coffey, and Ian Dunham) 58. Technical Notes for Sequencing Difficult Templates (David Stirling) 59. PCR-Based Detection of Nucleic Acidsin Chromosomes, Cells, and Tissues 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 (John H. Bull and Lynn Paskins) 61. Direct and Indirect In Situ PCR (Klaus Hermann Wiedorn and Torsten Goldmann) 62. Reverse Transcriptase In Situ PCR----New Methods in Cellular Interrogation (Mark Gilchrist and A. Dean Befus) 63. Primed In Situ Nucleic Acid Labeling Combined with Immunocytochemistry to Simultaneously Localize DNA and Proteins in Cells and Chromosomes (Ernst J. M. Speel, Frans C. S. Ramaekers, and Anton H. N. Hopman) 64. Cloning and Mutagenesis--- A Technical Overview (Helen Pearson and David Stirling) 65. Using T4 DNA Polymeraseto Generate Clonable PCR Products (Kai Wang) 66. A T-Linker Strategy for Modificationand Directional Cloning of PCR Products (Robert M. Horton, Raghavanpillai Raju, and Bianca M. Conti-Fine) 67. Cloning Gene Family Members Using PCRwith Degenerate Oligonucleotide Primers (Gregory M. Preston) 68. cDNA Libraries from a Low Amount of Cells (Philippe Ravassard, Christine Icard-Liepkalns, Jacques Mallet,and Jean Baptiste Dumas Milne Edwards) 69. Creation of Chimeric Junctions, Deletions, and Insertions by PCR (Genevieve Pont-Kingdon) 70. Recombination and Site-Directed MutagenesisUsing Recombination PCR (Douglas H. Jones and Stanley C. Winistorfer) 71. Megaprimer PCR---- Application in Mutagenesis and Gene Fusion (Emily Burke and Sailen Barik)