In 1985, Kary Mullis was driving late at night through the flowering buckeye to the ancient California redwood forest, cogitating upon new ways to sequence DNA. Instead he came upon a way to double the number of specific DNA modules, and to repeat the process essentially indefinitely. 1 He thought of using two oligonucleotide sequences, oppositely oriented, and a DNA polymerase enzyme, to double the number of DNA targets. Each product would thene become the target for the next reaction, effectively yielding a product which doubled in quantity with each repeated cycle. Like the chain reaction leading to nuclear fission, with each cycle event each initial reactant (neutron or DNA molecule) yields two similar products, each of which can serve as the initial reactant. The invention of this exponentially increasing amplification system quickly became known as the polymerase chain reaction (PCR). The tremendous sensitivity of PCR ultimately resides in the necessity for each of two specific oligonucleotide annealing reactions to occur at the same time in the proper orientation. The DNA annealing reaction is a very specific reaction. Single genes have been detected by hybridization of a DNA probe to chromosome preparations together with sensitive fluorescence microscopy. This is the equivalent to detecting a gene present in a single copy per cell genome. It is the combination of two such specific annealing reactions which makes possible the amplification needed to detect a single molecule with a specific DNA sequence in over 100,000 cell genomes.

Klappentext

The "how-to" book for every laboratory using PCR! Completeinformation aboutPCR procedures, combined with thediscussion of contemporary opinions, givesa good overviewof this multifaceted method and its applications.Even experienced laboratories will welcome the clearpresentation of demanding procedures such as the exactquantification of PCR products or the in-situ PCR. Protocolsthat have been tried and tested provide swift support fortroubleshooting all sorts of procedural problems. Indeed,many examples of problem areas found in diagnosticallyoriented PCR are fully described. Furthermore, the mostimportant lab PCR equipment as well as the pros and cons ofsingle individual reaction components which are necessaryfor performing PCR reactions are discussed.The book is clearly organized to give an introduction toeach technique, brief background information, step-by-stepinstructions for carrying out the procedure and additionalcomments.

Inhalt

1. PCR: principles and reaction components.- 1.1. PCR: A cyclic, exponential, in vitro amplification process.- 1.2. Temperature and time profile of thermal cycling.- 1.3. Taq DNA polymerase and reaction buffer.- 1.4. Deoxynucleotide triphosphates (dNTPs).- 1.5. Primers.- 1.6. Oil overlay and reaction volume.- 1.7. DNA sample.- 1.8. Size and structure of amplification products.- 1.9. PCR set-up strategies.- 2. Optimization strategies.- 2.1. Analytical PCR.- 2.2. Preparative PCR.- 3. General applications of PCR.- 3.1. Asymmetric PCR.- 3.2. Allele-specifîc amplification (ASA).- 3.3. Nested PCR.- 3.4. Multiplex PCR.- 3.5. Differential PCR.- 3.6. Competitive PCR.- 3.7. Amplification of unknown sequences.- 3.8. Application of PCR for genetic examinations, DNA amplification fingerprinting.- 3.9. Amplification with consensus primers.- 3.10. Expression PCR.- 3.11. Detection of infectious agents or rare sequences.- 4. Substances affecting PCR: Inhibition or enhancement.- 4.1. Inhibition introduced by native material.- 4.2. Inhibition introduced by reagents during DNA isolation: Detergents, Proteinase K and Phenol.- 4.3. Additives influencing the efficiency of PCR.- 4.4. Evaluation of the effect of individual additives.- 5. PCR: Contamination and falsely interpreted results.- 5.1. Control experiments.- 5.2. Sources of contaminations.- 5.3. Prevention and elimination of contamination.- 6. Biological material amenable to PCR.- 6.1. Sample preparation and storage.- 7. Isolation of DNA from cells and tissue for PCR.- 7.1. Alkaline lysis.- 7.2. Guanidinium rhodanid method (GuSCN).- 7.3. Proteinase K digestion for the isolation of DNA.- 7.4. Automated DNA extraction.- 8. Isolation of RNA from cells and tissue for PCR.- 8.1. General preparations.- 8.2. Inhibition of endogenous RNases.-8.3. Methods for the isolation of RNA suitable for RT-PCR.- 9. Reverse transcription/PCR (RT-PCR).- 9.1. Setting up an RT-PCR.- 9.2. Selective RT-PCR.- 10. Methods for identification of amplified PCR products.- 10.1. Agarose gel electrophoresis.- 10.2. DNA blot transfer and nonradioactive hybridization /detection.- 10.3. Detection systems of digoxigenin labeled hybrids.- 10.4. Polyacrylamide gel (PAGE) electrophoresis.- 11. Restriction fragment analysis.- 11.1. Restriction endonucleases.- 11.2. Endonuclecase selection.- 11.3. Optimal digestion conditions.- 12. Multiplex PCR.- Method: Detection of three different mycobacterial genom regions in a single PCR tube.- 13. Detection of single base changes using PCR.- 13.1. Allele-specific amplification (ASA, PASA, ASP, ARMS).- 13.2. Allele-specific oligonucleotide hybridisation (ASO).- 13.3. Chemical mismatch cleavage method.- 13.4. Denaturing gradient gel electrophoresis (DGGE).- 13.5. PCR single-strand conformation polymorphism (PCR-SSCP).- 14. Non-radioactive, direct, solid-phase sequencing of genomic DNA obtained from PCR.- 14.1. Generation of single-stranded DNA fragments.- 14.2. Sequencing of biotinylated PCR products.- 15. Application of PCR to analyze unknown sequences.- 15.1. Inverse PCR.- 15.2. Alternative methods to inverse PCR.- 16. Quantification of PCR-products.- 17. Cloning methods using PCR.- Method: Cloning and purification of a partial gag-sequence of HIV-1 using PCR.- 18. Site-directed mutagenesis using the PCR.- 19. In-situ polymerase chain reaction.- 20. Oligonucleotides in the field of PCR.- 20.1. Guidelines for designing PCR primers.- 20.2. Synthesis of primers.- 20.3. Purification of oligonucleotides.- 20.3. Chemical modification of primers.- 21. Review of different heat-stable DNA polymerases.- 21.1.Taq DNA polymerase.- 21.2. Vent polymerase (Thermococcus litoralis).- 21.3. Thermus thermophilus DNA polymerase.- 21.4. Pfu DNA polymerase (Pyrococcus furiosus).- 21.5. Bst polymerase.- 21.6. Fidelity of different heat-stable DNA polymerases.- 22. Physical features of thermocyclers and of their influence on the efficiency of PCR amplification.- 23. Alternative methods to PCR.- 23.1. Ligase chain reaction (LCR).- 23.2. Transcription-based amplification system (TAS).- 23.3. Self-sustained sequence replication (3SR).- 23.4. Q-beta replicase.- Addendum: Methodological examples for the application of the Polymerase chain reaction.- A) Characterization of oncogenes.- B) Detection of infectious agents.- C) Basic methodology and research applications.- Appendix I: Suppliers of specialist items.- Appendix II: List of Contributors.- Appendix III: DNA sequencing chrornato grams.