Main description:

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.

Contents:

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.- 1.2.1. Initial denaturation.- 1.2.2. Primer annealing.- 1.2.3. Primer extension.- 1.2.4. Denaturation step.- 1.2.5. Cycle number.- 1.2.6. Final extension.- 1.3. Taq DNA polymerase and reaction buffer.- 1.4. Deoxynucleotide triphosphates (dNTPs).- 1.5. Primers.- 1.5.1. Primer-related PCR artifacts.- 1.5.2. Melting temperature (Tm) and optimized annealing temperature.- 1.5.3. Primer concentration.- 1.6. Oil overlay and reaction volume.- 1.7. DNA sample.- 1.7.1. Purity of samples.- 1.7.2. Homogeneity of samples.- 1.7.3. DNA content of samples.- 1.8. Size and structure of amplification products.- 1.9. PCR set-up strategies.- Method: Mastermix PCR.- 2. Optimization strategies.- 2.1. Analytical PCR.- 2.1.1. Specificity.- 2.1.2. Product analysis (agarose gel) and trouble-shooting.- 2.1.3. Sensitivity.- 2.2. Preparative PCR.- 2.2.1. Reamplification.- 2.2.2. Incorporation of label using Taq polymerase.- Method 1: Reamplification of PCR products 29 Method 2: PCR synthesis of double- and single-stranded probes.- 3. General applications of PCR.- 3.1. Asymmetric PCR.- 3.2. Allele-specific 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.- 4.1.1. Blood.- 4.1.2. Fixed paraffin-embedded tissue.- 4.2. Inhibition introduced by reagents during DNA isolation: Detergents, Proteinase K and Phenol.- 4.2.1. Detergents.- 4.2.2. Proteinase K.- 4.2.3. Phenol.- 4.2.4. Salts and buffer systems.- 4.3. Additives influencing the efficiency of PCR.- 4.3.1. Dimethyl sulfoxide (DMSO).- 4.3.2. Glycerol.- 4.3.3. Formamide.- 4.3.4. Polyehtylene glycol (PEG).- 4.3.5. TWEEN.- 4.4. Evaluation of the effect of individual additives.- Method: Improving the efficiency of PCR amplification of HIV-1-provirus DNA from patient samples using a panel of additives.- 5. PCR: Contamination and falsely interpreted results.- 5.1. Control experiments.- 5.2. Sources of contaminations.- 5.2.1. Pre-PCR contaminations.- 5.2.2. Post-PCR contaminations.- 5.3. Prevention and elimination of contamination.- 5.3.1. Laboratory architecture.- 5.3.2. Autoclave.- 5.3.3. Working place and equipment.- 5.3.4. Influence of contamination risks on experimental design.- 5.3.5. Decontamination procedures.- 5.3.6. Chemical modification of PCR products to prevent carryover.- 6. Biological material amenable to PCR.- 6.1. Sample preparation and storage.- 6.1.1. Peripheral blood leucocytes.- Method 1: Selective lysis of red blood cells.- Method 2: Buffy coat.- Method 3: Isolation of mononuclear cells using FICOLL-PAQUE (TM).- 6.1.2. Pharyngeal and mouthwashes.- 6.1.3. Sputum, bronchoalveolar fluids.- Method 4: Preparation of mycobacterial DNA from sputum.- 6.1.4. Cerebrospinal fluid.- 6.1.5. Urine.- 6.1.6. Plasma, serum and other samples without host cellular DNA.- 6.1.7. Biopsies and solid tissue samples.- 6.1.8. Formalin-fixed, paraffin-embedded tissue.- 7. Isolation of DNA from cells and tissue for PCR.- 7.1. Alkaline lysis.- Method 1: Isolation of DNA using alkaline lysis.- 7.2. Guanidinium rhodanid method (GuSCN).- 7.3. Proteinase K digestion for the isolation of DNA.- Method 2: Proteinase K/phenol-chloroform extraction.- Method 3: PCR-adapted proteinase K method for leucocytes or whole blood.- Method 4: DNA isolation from paraffin-embedded tissue.- Method 5: Detection of viral DNA from serum.- 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.- Method 1: Acid guanidinium thiocyanate-phenol-chloroform extraction.- Method 2: Microadaption of the guanidinium-thiocyanate/CsCl ultracentrifugation method.- Method 3: Quick RNA isolation.- 9. Reverse transcription/PCR (RT-PCR).- 9.1. Setting up an RT-PCR.- 9.2. Selective RT-PCR.- Method 1: RNase-free DNase treatment of RNA samples.- Method 2: Reverse transcriptase using MoMLV-RT.- 10. Methods for identification of amplified PCR products.- 10.1. Agarose gel electrophoresis.- 10.1.1. Electrophoresis equipment.- 10.1.2. Gel loading buffer (GLB).- 10.1.3. UV-illumination and photography of stained gels.- 10.1.4. Molecular weight markers and semiquantitative gel analysis.- Method 1: Agarose gel casting.- Method 2: Post-PCR sample loading.- 10.2. DNA blot transfer and nonradioactive hybridization /detection.- 10.2.1. Capillary transfer (Southern blot).- Method 3: Southern blot from agarose gels.- 10.2.2. Dot blot and slot blot procedures.- Method 4: Dot blot protocol.- 10.2.3. Hybridization with digoxigenin-11-dUTP labeled DNA probes.- Method 5: Membrane hybridization procedure.- 10.3. Detection systems of digoxigenin labeled hybrids.- Method 6: Chromogen detection (NBT/BCIP).- Method 7: Chemiluminescence detection (AMPPD (TM)).- 10.4. Polyacrylamide gel (PAGE) electrophoresis.- 10.4.1. Separation of radioactive labeled amplification products and autoradiography.- Method 8: Vertical denaturing Polyacrylamide gel.- 10.4.2. Detection of non-radioactive amplification products in PAGE using silver stain.- Method 9: Silver stain of denaturing PAGE.- 11. Restriction fragment analysis.- 11.1. Restriction endonucleases.- 11.2. Endonuclecase selection.- 11.3. Optimal digestion conditions.- Method: Restriction digest of PCR products.- 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).- Method 1: Oligonucleotide hybridization using TMAC1 buffer.- 13.3. Chemical mismatch cleavage method.- Method 2: Chemical cleavage of mismatches in PCR products.- 13.4. Denaturing gradient gel electrophoresis (DGGE).- Method 3: Denaturing gradient gel elctrophoresis.- 13.5. PCR single-strand conformation polymorphism (PCR-SSCP).- Method 4: Sngle-strand conformation polymorphism (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.- Method 1: Manual sequencing procedure.- Method 2: Automatic sequencing procedure.- 15. Application of PCR to analyze unknown sequences.- 15.1. Inverse PCR.- Method 1: Inverse PCR.- 15.2. Alternative methods to inverse PCR.- 15.2.1. "Alu-PCR".- 15.2.2. Targeted gene walking PCR.- 15.2.3. "Panhandle PCR".- 15.2.4. Rapid amplification of cDNA ends (RACE-PCR).- Method 2: Rapid amplification of 3?cDNA ends (3?-RACE).- Method 3: Rapid amplification of 5?cDNA ends (5?-RACE).- 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.- Method 1: Purification of oligonucleotides by gel electrophoresis.- Method 2: Purification of oligonucleotides by reverse-phase HPLC.- Method 3: Quick purification of oligonucleotides using anion-exchange.- columns (01igoPak (TM)).- 20.3. Chemical modification of primers.- Method 4: Labelling of oligonucleotides by T4polynucleotide kinase.- Method 5: Chemical labelling of oligonucleotides using photoactive biotin.- Method 6: 3?-end biotin labelling by terminal deoxynucleotidyl transferase.- Method 7: Modifications of the 3?terminus with a primary aliphatic amine.- Method 8: 5?-end labelling by an amino linker during synthesis.- Method 9: 5?-end labelling by introduction of a sticky-and restriction site in the PCR with subsequent incorporation by Klenow polymerase.- 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).- Method: Amplification of a 365bp HIV-1-fragment using Pfu polymerase and influence of DMSO on amplification efficiency.- 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.- Detection of mutations at codon 61 of the c-Ha-ras gene in small precancerous liver lesions of the C3H mouse R Bauer-Hofmann, A Buchmann, F Klimek, M Schwarz.- Isolation and direct sequencing of PCR-cDNA fragments from tissue biopsies H Klocker, F Kaspar, J Eberle, G Bartsch.- Differential PCR: Loss of the ?1-interferon gene in chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL) A Neubauer, C Schmidt, B Neubauer, W Siegert, D Huhn, E Liu.- B) Detection of infectious agents.- Long-term persistence of Borrelia burgdorferi in neuroborreliosis detected by polymerase chain reaction S Bamborschke, A Kaufhold, A Podbielski, B Melzer, A Porr, B Rehse-Kupper.- Two-stage polymerase chain reaction for the identification of Borrelia burgdorferi in the tertiary stage of neuroborreliosis H Bocklage, R Lange, H Karch, J Heesemann, HW Koelmel.- Screening for CMV infection following bone marrow transplantation using the PCR technique H Einsele, M Steidle, M Muller, G Ehninger, JG Saal, CA Muller.- Detection of spumaviral sequences by polymerase chain reaction W Muranyi, R M Flugel.- The use of PCR for epidemiological studies of HNANB viruses in arthropod vectors R Seelig, CF Weiser, HW Zentraf, C Bottner, HP Seelig, M Renz.- C) Basic methodology and research applications.- In vitro amplification and digoxigenin labelling of single-stranded and double-stranded DNA probes for diagnostic in situ hybridization U Finckh, P A Lingenfelter, K W Henne, C Schmidt, W Siegert, D Myerson.- Differentiation of arylsulfatase A deficiencies associated with metachromatic leukodystrophy and arylsulfatase A pseudodeficiency V Gieselmann.- Molecular genetics of neuromuscular diseases - the role of PCR in diagnostics and research B Kadenbach, P Seibel.- The application of polymerase chain reaction for studying the phylogeny of bacteria G Koehler, W Ludwig, KH Schleifer.- Site-directed mutagenesis facilitated by PCR O Landt, U Hahn.- Ectopic transcription in the analysis of human genetic disease J Reiss.- Appendix I: Suppliers of specialist items.- Appendix II: List of Contributors.- Appendix III: DNA sequencing chrornato grams.