CONTENTS Contributors ... xi Preface ... xvii Part One. BASIC METHODOLOGY 1. Optimization of PCRs / Michael A. Innis ; David H. Gelfand ... 3 2. Amplification of Genomic DNA / Randall K. Saiki ... 13 3. Amplification of RNA / Ernest S. Kawasaki ... 21 4. RACE : Rapid Amplification of cDNA Ends / Michael A. Frohman ... 28 5. Degenerate Primers for DNA Amplification / Teresa Compton ... 39 6. cDNA Cloning Using Degenerate Primers / Cheng Chi Lee ; C. Thomas Caskey ... 46 7. PCR with 7-Deaza-2-Deoxyguanosine Triphosphate / Michael A. Innis ... 54 8. Competitive PCR for Quantitation of mRNA / Gary Gilliland ; Steven Perrin ; H. Franklin Bunn ... 60 9. Quantitativ ePCR / Alice M. Wang ; David F. Mark ... 70 10. Production of Single-Stranded DNA by Asymmetric PCR / Peter C. McCabe ... 76 11. Cloning with PCR / Stephen J. Scharf ... 84 12. Oligonucleotide Ligation Assay / Ulf Landegren ; Robert Kaiser ; Leroy Hood ... 92 13. Nonisotopically Labeled Probes and Primers / Corey Levenson ; Chu-an Chang 14. Incorporatin of Biotinylated dUTP / Y. -M. Dennis Lo ; Wajahat Z. Mehal ; Kenneth A. ... 113 15. Nonisotopiec Detection of PCR Products / Rhea Helmuth ... 119 16. Thermostable DNA Polymerases / David H. Gelfand ; Thomas J. White ... 129 17. Procedures to Minimize PCR-Product Carry-Over / Shirley Kwok ... 142 18. Sample Preparation from Blood, Cells, and Other Fluids / Ernest S. Kawasaki ... 146 19. Sample Preparation from Paraffin-Embedded Tissues / Deann K. Wright ; M. Michele Manos ... 153 20. Amplifying Ancient DNA / Svante P a · a ·· bo ... 159 Part Two. RESEARCH APPLICATIONS 21. In Vitro Transcription of PCR Templates / Michael J. Holland ; Michael A. Innis ... 169 22. Recombinant PCR / Russell Higuchi ... 177 23. DNase I Footprinting / Barbara Krummel ... 184 24. Sequencing with Taq DNA P9olymerase / Mary Ann D. Brow ... 189 25. Direct Sequencing with the Acid of Phage Promoters / Steve S. Sommer ; Gobinda Sarkar ; Dwight ; D. Ko ... 197 26. Identifying DNA Polymorphisms by Denaturing Gradient Gel Electrohphresis / Val C. Sheffield ; David R. Cox ; Richard M. Myer ... 206 27. Amplification of Flanking Sequences by Inverse PCR / Howard Ochman ; Meetha M. Medhora ; Dan Garza ; D ... 219 28. Detection of Homologous Recombinants / Micahel A. Frohman ; Gail R. Martin ... 228 29. RNA Processing : Apo-B / Lyn M. Powell ... 237 30. A Transcription-Based Amplification System / T. R. Gingeras ; G. R. Davids K. M. Whitfiled ; H ... 245 31. Screening of λgt11 Libraries / Kenneth D. Fiedman ; Nancy L. Rosen ; Peter J. Ne ... 253 Part Three. GENETICS AND EVOLUTION 32. HLA DNA Typing / Henry A. Erlich ; Teodorica L. Bugawan ... 261 33. Multiplex PCR for the Diagnosis of Duchenne Muscular Dystrophy / Jeffrey S. Chamberlain ; Richard A. Gibbs ; Joel ... 272 34. Isolation of DNA from Fungal Mycelia and Single Spores / Steven B. Lee ; John W. Taylor ... 282 35. Genetic Prediction of Hemophilia A / Scott C. Kogan ; Jane Gitschier ... 288 36. Haplotype Analysis from Single Sperm or Diploid Cells / Ulf Gyllensten ... 300 37. Amplification of Ribosomal RNA Genes for Molecular Evolution Studies / Mitchell L. Sogin ... 307 38. Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics / T. J. White ; T. Bruns ; S. Lee ; J. Taylor ... 315 Part Four. DIAGNOSTICS AND FORENSICS 39. Detection of Human T-Cell Lymphoma/Leukemia Viruses / Garth D. Ehrlich ; Steven Greenberg ; Mark A. Abb ... 325 40. Detection of Human Immunodeficiency Virus / David E. Kellogg ; Shirley Kwok ... 337 41. Detection fo Hepatities B Virus ... 348 42. Detection and Typing of Genital Human Papillomaviruses / Yi Ting ; M. Michele Manos ... 356 43. Detection of Human Cytomegalovirus / Darryl Shibata ... 368 44. PCR Amplification of Enteroviruses / Harley A. Rotbart ... 372 45. Novel Viruses / David Mack ; Oh-Sik Kwon ; Fred Fallona ... 378 46. Analysis of ras Gene Point Mutations by PCR and Oligonucelotide Hybridization / John Lyons ... 386 47. B-Cell Lymhpoma : t(14,18) Chromosome Rearrangement / Marco Crescenzi ... 392 48. Detecting Bacterial Pathogens in Environmental Water Samples by Using PCR and Gene Probes / Ronald M. Atlas ; Asim K. Bej ... 399 49. PCR in the Diagnosis of Retinoblastoma / Sang-Ho Park ... 407 50. Determination of Familial Relationships / Cristi a n Orrego ; Mary Chaire King ... 416 Part Five. INSTRUMENTATION AND SUPPLIES 51. PCR in a Teacup : A Simple and Inexpensive Method for Thermocycling PCRs / Robert Watson ... 429 52. A Low-Cost Air-Driven Cycling Oven / Peter Denton ; H. Reisner ... 435 53. Modification of a Histokinette for Use as an Automated PCR Machine / N. C. P. Cross ; N. S. Foulkes ; D. Chappel ; J. ... 442 54. Organizing a Laboratory for PCR Work / Cristian Orrego ... 447 55. Basic Equipment and Supplies / Roberta Madej ; Stephen Scharf ... 455 Index ... 461