COMPARATIVE BIOCHEMISTRY: SUPPLEMENT
Chapter 1 Introduction to Biochemistry and Molecular Biology 1 Introduction 1 11 Biochemical and Molecular Biology 2 111 Protein Structure and Function 4 112 Metabolism and Metabolic Pathways 6 113 Nucleic Acids and Gene Expression 7 114 Cellular Signaling and Communication9 115 Genetic Engineering and Recombinant DNA Technology 11 116 Bioinformatics and Computational Biology 12 117 Cellular and Molecular Immunology 14 12 Unit of Measurement in Biochemical and Molecular Biology 16 121 Spectroscopy Units 17 122 Gel Electrophoresis19 123 Enzyme Kinetics 22 124 Protein Concentration 23 125 Nucleic Acid Amplification 25 13 Weak Electrolytes 26 131 Characteristics of Weak Electrolytes 27 132 Ionization Constants and Equilibrium 29 133 pH and Weak Electrolytes 30 TABLE OF CONTENTS 134 Biological Importance of Weak Electrolytes 32 135 Titration and Weak Electrolytes 33 136 Comparison with Strong electrolytes 35 137 Factors Affecting the Extent of Ionization36 References 38 Chapter 2 Cell Culture Techniques and Centrifugation 39 Introduction 39 21 Centrifugation Techniques-Basic Principles, Analytical and Preparative Centrifugation, their Applications 40 211 Principle 40 212 Application of Centrifugation43 213 Applications of Cell Culture 44 214 Spectrophotometry: UV-Visible Spectrophotometer 49 215 Column chromatography – An Example of The Equipment Used in Low-Performance Liquid 51 216 High-Performance Liquid Chromatography (HPLC) 52 217 Electrophoresis: Theory and Different Types – Page, SDS-Page, Capillary Electrophoresis, and IEF 54 22 Molecular Biology Techniques-Southern Blotting, Northern Blotting, Western Blotting, Microarray Technology 58 221 Complementation Techniques; Polymerase Chain Reaction (PCR), Molecular Markers and Plant Diversity60 222 NON-PCR based Approaches67 223 PCR Based Approaches 68 224 Amplified Fragment Length Polymorphism (AFLP) 69 225 DNA Polymorphism using PCR 70 226 Sequence Tagged Microsatellite Sites (STMS) or SSR loci 72 227 Plant diversity 73 References 74 Chapter 3 Basic Molecular Biology Techniques 75 Introduction 75 31 Structure and Function of Nucleic Acids 75 311 The Structure of Deoxyribonucleic Acid 76 312 DNA Replication 82 32 Enzymes Used In Molecular Biology 88 33 Isolation And Separation Of Nucleic Acids 90 331 Isolation of DNA 90 332 Isolation of RNA 92 34 Electrophoresis of Nucleic Acids 93 35 Restriction Mapping of DNA Fragments 95 36 Nucleic Acid Analysis Methods 95 361 DNA Blotting 96 362 RNA Blotting 97 37 Gene Probe Derivation 98 38 Labelling DNA Gene Probe Molecules 99 381 End Labelling of DNA Molecules 100 382 Random Primer Labelling 101 383 Nick Translation 102 39 Polymerase Chain Reaction 103 References 105 Chapter 4 Recombinant DNA and Genetic Analysis 107 Introduction 107 41 Constructing Gene Libraries 108 411 Digesting Genomic DNA Molecules 108 412 Ligating DNA Molecules 109 413 Aspects of Gene Libraries 111 414 Genomic DNA Libraries 112 415 cDNA Libraries 113 416 Treatment of Blunt cDNA Ends 115 417 Enrichment Methods for RNA 116 418 Subtractive Hybridization 116 419 Cloning PCR Products 117 42 Cloning Vectors 118 421 Plasmids 119 422 Virus-based Vectors 123 423 M13 and Phagemid-based Vectors 128 424 Cosmid Based Vectors 129 425 Large Insert Capacity Vectors 130 426 Yeast Artificial Chromosome (YAC) Vectors 130 References 131 Chapter 5 Immunochemical Techniques 133 Introduction 133 51 Overview of Immunochemical Techniques 134 511 Characteristics and Role 134 52 Methods of Analysis 135 521 Antigens 136 522 Antibodies 137 53 Precipitation Methods in Gel 138 531 Agglutination 141 532 Immunoprecipitation 143 533 Immunoelectrophoresis 144 534 Immunofixation 145 535 Immunoturbidimetry 145 536 Immunonephelometry 146 537 Immunoassay 146 538 Competitive Binding 147 54 Fluorescence Activated Cell Sorting 147 541 Fluorescence Activated Cell Sorting Protocol 149 542 Applications of Fluorescence Activated Cell Sorting 149 55 Immunoaffinity Chromatography 150 551 Structure and Properties of Antibodies 151 552 Production of Antibodies 153 553 Supports for IAC155 554 Antibody-immobilization Methods 157 555 Chromatographic Separation 159 556 Application and Elution Conditions 162 References 163 Chapter 6 Protein Structure, Purification, Characterisation and Function Analysis 165 Introduction 165 61 Properties of Amino Acids and Proteins 165 611 Classification 166 612 Configuration 169 62 Protein Structure 170 621 Primary Structure 170 622 Secondary Structure 173 623 Tertiary Structure 174 624 Quaternary Structure 175 625 Denaturation and Protein Folding 176 63 Protein Purification 177 631 Extraction 177 632 Precipitation and Differential Solubilization 178 633 Ultracentrifugation 179 634 Purification Strategy 181 635 Purification Scheme 189 64 Protein Structure Determination 192 641 X-Ray Crystallography 192 642 Electron Microscopy and Cryo-Electron Microscopy 193 643 Nuclear Magnetic Resonance (NMR) Spectroscopy 193 644 Small-Angle X-Ray Scattering and Small-Angle Neutron Scattering 193 645 Homology Modeling 193 646 Partial Structural Study Methods 194 65 Proteomics and Protein Function 194 References 196 Chapter 7 Techniques Used in Biochemistry and Molecular Biology 199 Introduction 199 71 Mass Spectroscopic Techniques 200 711 Electrospray Ionization (ESI) 201 712 Matrix-Assisted Laser Desorption/Ionization (MALDI) 202 713 Tandem Mass Spectrometry 204 714 High-Resolution Mass Spectrometry (HRMS) 205 715 Quantitative Mass Spectrometry 206 716 Imaging Mass Spectrometry 207 72 Electrophoretic Techniques 209 721 Agarose Gel Electrophoresis 210 722 Polyacrylamide Gel Electrophoresis (PAGE) 211 723 Capillary Electrophoresis (CE) 212 724 Isoelectric Focusing (IEF) 214 725 Protein Blotting Techniques (Western Blotting) 215 726 DNA Gel Electrophoresis 216 727 RNA Gel Electrophoresis 217 73 Chromatographic Techniques 219 731 Reversed-Phase Chromatography 220 732 Size Exclusion Chromatography 221 733 Ion Exchange Chromatography 223 734 Hydrophobic Interaction Chromatography 224 735 Gas Chromatography225 736 High-Performance Liquid Chromatography (HPLC) 228 74 Spectroscopic Techniques 229 741 Ultraviolet-Visible Spectroscopy (UV-Vis) 231 742 Infrared Spectroscopy (IR) 232 743 Nuclear Magnetic Resonance (NMR) Spectroscopy 234 744 Fluorescence Spectroscopy 235 745 Raman Spectroscopy 239 References 240 Chapter 8 Principles of Clinical Biochemistry 243 Introduction 243 81 Principles of Clinical Biochemical Analysis 244 811 Basis of Analysis of Body Fluids for Diagnostic, Prognostic and Monitoring Purposes 244 812 Reference Ranges 248 82 Clinical Measurements and Quality Control 250 821 The Operation of Clinical Biochemistry Laboratories 250 822 Analytical Organization 253 823 Audit and Accreditation 260 83 Examples of Biochemical Aids to Clinical Diagnosis 263 831 Principles of Diagnostic Enzymology 263 832 Ischaemic Heart Disease and Myocardial Infarction265 833 Liver Disease 269 834 Kidney Disease270 835 Endocrine Disorders 275 836 Hypothalamus–Pituitary–Gonad Axis 276 837 Diabetes Mellitus 276 838 Plasma Proteins 280 References 282