ISBN: 3-540-57129-9
TITLE: Techniques in Molecular Medicine
AUTHOR: Hildebrandt, Friedhelm; Igarashi, Peter (Eds.)
TOC:

Part I Molecular Biology Techniques 
Chapter 1 
Structure and Function of the Human Genome 
FRIEDHELM HILDEBRANDT 3 
Chapter 2 
Enzymatic Modification of DNA and RNA 
KAI-OLAF NETZER 24 
Subprotocol 1: Restriction Endonucleases 24 
Subprotocol 2: Exonucleases, Other Nucleases and Ligases 33 
Subprotocol 3: Phosphatases - CIP Reaction 36 
Subprotocol 4: Kinases 37 
Subprotocol 5: DNA-Dependent DNA Polymerases - 
E. Coli DNA Polymerase 1 and Klenow Enzyme 40 
Subprotocol 6: T4 and T7 DNA Polymerase 42 
Subprotocol 7: RNA-Dependent DNA Polymerases 44 
Subprotocol 8: DNA - Dependent RNA Polymerases 47 
Subprotocol 9: Terminal Deoxynucleotidyl Transferase (TdT) 49 
Chapter 3 
Purification and Nucleic Acids From Eukaryotic Cells 
HANS GERD NOTHWANG AND FRIEDHELM HILDEBRANDT 50 
Subprotocol 1: Extraction of Nucleic Acids 50 
Subprotocol 2: Isolation of Genomic DNA From Mammalian Cells 56 
Subprotocol 3: Isolation of Cytoplasmic RNA 60 
Subprotocol 4: Rapid Preparation of Total RNA by Acid 
Guanidinium Thiocyanate/Phenol/Chloroform/ Extraction 64 
Subprotocol 5: Preparation of Poly(A)+ RNA 67 
Chapter 4 
Electrophoresis of DNA and RNA Fragments 
FENG QIAN AND GREGORY G. GERMINO 71 
Subprotocol 1: The Recovery of DNA Fragments 75 
Subprotocol 2: Electrophoresis of RNA 77 
Chapter 5 
Single-Strand Conformation Polymorphism (SSCP) Analysis 
FENG QIAN AND GREGORY G. GERMINO 83 
Chapter 6 
Denaturing Gradient Gel Electrophoresis 
KAI-OLAF NETZER 86 
Chapter 7 
Introduction of Pulsed-Field Gel Electrophoresis (PFGE) 
FENG QIAN AND GREGORY G. GERMINO 96 
Chapter 8 
Pulsed-Field Gel Electrophoresis: Protocols 
GUDRUN A. RAPPOLD, KARIN RIED, ALBRECHT KLINK, 
ERCOLE RAO AND BIRGIT WEISS 103 
Subprotocol 1: Preparation and Processing of High Molecular 
DNA in Agarose Blocks 104 
Subprotocol 2: Preparation of Size Markers 106 
Subprotocol 3: Restriction Enzyme Digestion of DNA 
in Agarose Blocks 108 
Subprotocol 4: Gel Electrophoresis 110 
Chapter 9 
Radioactive Labeling of DNA and RNA Probes 
PETER IGARASHI 114 
Subprotocol 1: End-Labeling Using T4 Polynucleotide Kinase 114 
Subprotocol 2: End-Labeling Using the Exchange Kinase Reaction 116 
Subprotocol 3: Labeling 3' Ends with Terminal Transferase 
(Forward Reaction) 117 
Subprotocol 4: Random-Prime Labeling 118 
Subprotocol 5: Riboprobes 121 
Chapter 10 
Hybridization Methods (Southern and Northern Blotting) 
KAI-OLAF NETZER 126 
Subprotocol 1: Preparation of Dot Blots for DNA 129 
Subprotocol 2: Preparation of Southern Blots for Genomic DNA 131 
Subprotocol 3: Hybridization of DNA Blots 135 
Subprotocol 4: Preparation of RNA Dot Blots 139 
Subprotocol 5: Preparation of Northern Blots 140 
Subprotocol 6: Hybridization of RNA Blots 143 
Subprotocol 7: Membrane Stripping 147 
Chapter 11 
Specimen Preparation Techniques for Cytogenetic Analysis 
by Fluorescence in situ Hybridization 
STEFAN JOOS, MARTIN BENTZ, ANTON H. N. HOPMAN 
AND PETER LICHTER 148 
Subprotocol 1: Cultivation of Blood Lymphocytes 149 
Subprotocol 2: Preparation of Metaphase Chromosomes 
and Interphase Nuclei from Blood Lymphocytes 151 
Subprotocol 3: Pepsin Digestion of Blood Lymphocytes 153 
Subprotocol 4: Preparation of Fibroblasts for FISH 
(Adherently Growing Cells) 154 
Subprotocol 5: Treatment of Blood Smears 155 
Subprotocol 6: Bone Marrow Smears 156 
Subprotocol 7: Clinical Material Obtained from Solid Tumors: 
Pretreatment of Slides with Poly-L-Lysine 157 
Subprotocol 8: Preparation of Single Cell Suspensions 
from Solid Tumor Material 158 
Subprotocol 9: Preparation of Frozen Sections 160 
Subprotocol 10: Paraffin-Embedded Tissue Sections 161 
Chapter 12 
Chromosome Analysis by Fluorescence in situ Hybridization 
MARTIN BENTZ, STEFAN JOOS AND PETER LICHTER 166 
Subprotocol 1: Specimen Preparation 170 
Subprotocol 2: Probe Labelling by Nick Translation 171 
Subprotocol 3: Dot-Blot Assay to Test Labelling 174 
Subprotocol 4: Probe Mixture and Denaturation for Fluorescence 
in situ Hybridization 175 
Subprotocol 5: Denaturation of the DNA on the Slides 176 
Subprotocol 6: Detection 178 
Subprotocol 7: Chromosomal in situ Suppression (CISS) 
Hybridization 179 
Subprotocol 8: Signal Amplification 181 
Subprotocol 9: Multicolor Fluorescence in situ Hybridization 182 
Chapter 13 
Sequencing 
KLAUS DEICHMANN 186 
Subprotocol 1: Standard protocol for T7 DNA polymerase 
(Sequenase(TM)) 194 
Subprotocol 2: Standard Protocol for Sequencing with Taq 
Polymerase 197 
Subprotocol 3: Denaturing Polyacrylamide Sequencing Gels 200 
Chapter 14 
Polymerase Chain Reaction 
FRIEDHELM HILDEBRANDT AND IVA SINGH-SAWHNEY 207 
Chapter 15 
Cloning Vectors 
ANNETTE DEICHMANN AND KLAUS DEICHMANN 226 
Subprotocol 1: Transformation of Bacteria with Recombinant 
Plasmid Using the Ca++-Precipitation Method 
(Mandel 1970) 234 
Subprotocol 2: Transformation of Bacteria with Recombinant 
Plasmid by Electroporation (Dower 1988) 238 
Subprotocol 3: Transformation of Bacteria with Recombinant 
Bacteriophage DNA 241 
Subprotocol 4: "Mini-Prep" Method for Plasmid Isolation 245 
Subprotocol 5: Chromatographic Method for Plasmid Preparation 247 
Subprotocol 6: Large-Scale Preparation of Bacteriophage DNA 
(Replicative Form (RF)) 249 
Part II Strategies for Gene Isolation and Characterization 
Chapter 16 
Cloning Strategies: An Overvierw 
FRIEDHELM HILDEBRANDT 257 
Chapter 17 
Genomic Libraries 
LUITZ F. ONUCHIC AND GREGORY G. GERMINO 261 
Chapter 18 
cDNA Libraries 
LUITZ F. ONUCHIC AND GREGORY G. GERMINO 273 
Chapter 19 
cDNA Library Screening 
JOHANN PETER HOSSLE 288 
Subprotocol 1: Plating of a lambda gt11 cDNA Library 296 
Subprotocol 2: Hybridization Screening - cDNA Sequences as Probes 298 
Subprotocol 3: Immuno-Screening 300 
Subprotocol 4: Purification of Phage Particles, DNA Isolation 
and Subcloning 306 
Chapter 20 
Expression Cloning with Xenopus Oocytes 
DANIEL MARKOVICH, ANDREAS WERNER AND HEINI MURER 310 
Chapter 21 
Transgenic Animals 
ECKART SCHOTT, MARTIN PAUL AND DETLEV GANTEN 319 
Chapter 22 
Expression in Eukaryotic Cells: Gene Transfer Using Retroviral Vectors 
MARTIN MARX 337 
Subprotocol 1: Synthesis of the Insert 339 
Subprotocol 2: Purification 340 
Subprotocol 3: Ligation of the Insert into the Vector 340 
Subprotocol 4: Transfection of E. coli HB101 and Plasmid 
DNA Isolation 341 
Subprotocol 5: Assay for the Presence of the DNA Insert 343 
Subprotocol 6: Transfection of Psi2 Packaging Cells 344 
Subprotocol 7: Infection of PsiAM Packaging Cells 348 
Subprotocol 8: Infection of Target Cells 348 
Chapter 23 
Positional Cloning and Linkage Analysis 
FRIEDHELM HILDEBRANDT AND HEYMUT OMRAN 352 
Part III Protein Methods 
Chapter 24 
Protein Techniques 
ROBERT F. REILLY 367 
Subprotocol 1: In Vitro Translation and Immunoprecipitation 367 
Subprotocol 2: Western Blotting 375 
Subject Index 379 
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