PCR: Clinical Diagnostics and Research

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-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.- 4.1.1. 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-PAQUET.- 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 (AMPPDT).- 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 (01igoPakT).- 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-Küpper.- 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 Kölmel.- Screening for CMV infection following bone marrow transplantation using the PCR technique H Einsele, M Steidle, M Müller, G Ehninger, JG Saal, CA Müller.- Detection of spumaviral sequences by polymerase chain reaction W Muranyi, R M Flügel.- 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 Köhler, 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.