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Practical Techniques in Molecular Biotechnology [Kõva köide]

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  • Formaat: Hardback, 378 pages, kõrgus x laius x paksus: 248x190x21 mm, kaal: 760 g, Worked examples or Exercises
  • Ilmumisaeg: 16-Jun-2022
  • Kirjastus: Cambridge University Press
  • ISBN-10: 1108486401
  • ISBN-13: 9781108486408
Teised raamatud teemal:
Practical Techniques in Molecular BiotechnologySuurem pilt
  • Formaat: Hardback, 378 pages, kõrgus x laius x paksus: 248x190x21 mm, kaal: 760 g, Worked examples or Exercises
  • Ilmumisaeg: 16-Jun-2022
  • Kirjastus: Cambridge University Press
  • ISBN-10: 1108486401
  • ISBN-13: 9781108486408
Teised raamatud teemal:
Püsilink: https://www.kriso.ee/db/9781108486408.html
Märksõnad:
Practical Techniques in Molecular Biotechnology intends to familiarise students with the basics of the well-known experiments of molecular biotechnology and related courses like chemical biotechnology and cell biology. The content of the book will be useful in strengthening the basic skills and help students to apply the concepts to real-world problems. This book emphasises important concepts like bioanalytical techniques, biochemical analysis of proteins, recombinant DNA, and protein technology etc. The text will help students to understand the theoretical aspects of the techniques and provide experience with hands-on techniques to demonstrate practical troubleshooting and data analysis. The text is supported with diagrams, data, summaries for the quick recap and appendices with useful protocols and calculation methods.

This book will familiarise students with the basics of some of the well-known experimental molecular biotechnology and related disciplines. The book intends to give balanced exposure to experimental techniques, procedures, and data analysis. The book will be useful for undergraduate students as a supplementary/reference text.

Muu info

The book will be useful for undergraduate students as a supplementary/reference text in the field of molecular biotechnology.
List of Figures xiii
List of Tables xix
Preface xxi
1 Introduction 1(19)
1.1 Biotechnology - Its Background and History
1(7)
1.2 Technology and Laboratory Practice
8(2)
1.3 Pedagogical Strategy of Biochemical Technology Practice
10(4)
1.4 Laboratory Safety
14(1)
1.5 Biosafety and Biosafety Levels
15(5)
2 Recombinant DNA and Protein Technology 20(31)
2.1 Introduction
20(1)
2.2 Cloning
21(1)
2.3 Types of Cloning Vectors
21(6)
2.3.1 Plasmid Vectors
21(2)
2.3.2 Bacteriophages or Phage Lambda
23(1)
2.3.3 Cosmids
24(1)
2.3.4 Yeast Artificial Chromosomes (YACs)
25(2)
2.4 Expression Systems
27(4)
2.4.1 Bacterial Expression Systems
28(1)
2.4.2 Yeast Expression Systems
29(1)
2.4.3 Baculovirus Expression Systems
29(1)
2.4.4 Mammalian Expression Systems
29(1)
2.4.5 Cell Free Expression Systems
30(1)
2.5 Promoters
31(5)
2.5.1 The Lac Promoter
32(1)
2.5.2 The tac and trc Promoters
32(1)
2.5.3 The ara Promoter
33(1)
2.5.4 The T7 Promoter
34(1)
2.5.5 The Lambda Promoters
35(1)
2.5.6 Cold-shock Promoters
35(1)
2.5.7 Non-promoter Regulatory Elements
35(1)
2.6 Protein Purification Methods
36(9)
2.6.1 Cell Disruption
38(1)
2.6.2 Purification Methods
39(1)
2.6.3 Affinity Purification
40(2)
2.6.4 Gel Filtration or Size Exclusion
42(1)
2.6.5 Salting In/Salting Out
42(2)
2.6.6 Analytical Centrifugation
44(1)
2.7 Monitoring Protein Purification
45(6)
2.7.1 Determination of Protein Concentration
45(1)
2.7.2 Ultraviolet Absorption
46(1)
2.7.3 Bradford Method
46(1)
2.7.4 Lowry (Folin-Ciocaltaeu) Method
46(1)
2.7.5 The Bicinchoninic Acid (BCA) Method
46(1)
2.7.6 Kjeldahl Method
47(4)
3 Enzyme Kinetics, Proteomics, and Mass Spectrometry 51(52)
3.1 Order and Molecularity
52(1)
3.2 Important Theories Related to Enzyme Kinetics
52(5)
3.2.1 Collision Theory
52(2)
3.2.2 Transition State Theory
54(2)
3.2.3 Arrhenius Equation
56(1)
3.3 Enzymes
57(4)
3.3.1 Catalytic Mechanism
58(1)
3.3.2 Enzyme Unit
58(1)
3.3.3 Transition States and Reaction Rates
59(1)
3.3.4 Initial Velocity
60(1)
3.4 Enzyme Kinetics
61(3)
3.4.1 Order of Reaction
61(1)
3.4.2 Michaelis-Menten Analysis
62(1)
3.4.3 The Significance of Km,kkatandkcat/Km
63(1)
3.5 Graphs of the Michaelis-Menten Equation
64(3)
3.5.1 Plotting v Against [ s]
64(1)
3.5.2 The Double-reciprocal Plot
64(1)
3.5.3 The Plot of [ S]/v Against [ S]
65(1)
3.5.4 The Plot of v Against v/a
65(1)
3.5.5 The Direct Linear Plot
66(1)
3.6 Enzyme Inhibition
67(5)
3.6.1 Competitive Inhibition
68(1)
3.6.2 Noncompetitive Inhibition
68(1)
3.6.3 Uncompetitive Inhibition
69(1)
3.6.4 Mixed Inhibition
70(2)
3.6.5 Irreversible Inhibition
72(1)
3.7 Inhibitory Effect of Substrates
72(2)
3.7.1 Non-productive Binding
72(1)
3.7.2 Substrate Inhibition
73(1)
3.8 General Protocol for an Inhibition Experiment
74(1)
3.9 Applications of Enzyme Inhibition
75(1)
3.10 Methodologies for Studying Catalytic Mechanism of the Enzyme
75(1)
3.11 Enzyme Activation
76(2)
3.11.1 Specific Activation
76(1)
3.11.2 Hyperbolic Activation and Inhibition
77(1)
3.12 Proteomics
78(6)
3.12.1 2-D Page
79(3)
3.12.2 Mass-fingerprinting
82(2)
3.13 Mass Spectrometry
84(19)
3.13.1 Introduction
84(19)
3.13.1.1 Principles
85(1)
3.13.1.2 Vacuum System
86(1)
3.13.1.3 Sample System
86(1)
3.13.1.4 Ion Source (ionization)
87(5)
3.13.1.5 Mass Analyzer
92(1)
3.13.1.6 Applications
92(2)
3.13.1.7 Identification and Sequence Determination of Peptides and Proteins
94(9)
4 Bioanalytical Techniques 103(66)
4.1 Introduction
103(2)
4.1.1 Requirements for Structure and Function of Biomolecules and Affecting Factors
104(1)
4.2 Hydrodynamic Methods
105(3)
4.2.1 Sedimentation
106(1)
4.2.2 Use of Sedimentation or Centrifugation Techniques
107(1)
4.3 Biocalorimetry
108(4)
4.4 Spectroscopic Techniques Used on Biomolecules
112(33)
4.4.1 Basis and Purpose
112(1)
4.4.2 Sigma (s) and Pi (p) Orbitals and Bonds
112(3)
4.4.3 Benzene and Aromatic Molecular Stabilities
115(1)
4.4.4 Molecular Structure and Transitions
116(9)
4.4.4.1 Conjugated p Electron System
116(1)
4.4.4.2 Molecular Features and Electronic Transitions
117(5)
4.4.4.3 Structure and Optical Properties of Biological Chromophores
122(3)
4.4.5 Ultraviolet/Visible Absorption Spectroscopy
125(11)
4.4.6 Fluorescence Spectroscopy
136(5)
4.4.7 Use of Fluorescence
141(4)
4.5 Circular Dichroism (CD)
145(6)
4.5.1 Spectral Characteristics
146(5)
4.6 FTIR Spectroscopy
151(3)
4.6.1 Analysis of Protein FTIR Data
153(1)
4.7 Electrophoretic Techniques
154(15)
4.7.1 Theory of Electrophoresis
154(3)
4.7.2 Polyacrylamide Gel
157(1)
4.7.3 Agarose Gels
157(1)
4.7.4 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
158(4)
4.7.4.1 Ion Mobility and Protein Stacking
160(1)
4.7.4.2 What Happens Once the Proteins Have Been Stacked?
160(1)
4.7.4.3 Buffer Systems Used in Gel Electrophoresis
161(1)
4.7.4.3.1 Continuous Buffer Systems
161(1)
4.7.4.3.2 Discontinuous Buffer Systems
161(1)
4.7.4.4 Safety
162(1)
4.7.5 Isoelectric Focusing (IEF) Gel Electrophoresis
162(3)
4.7.5.1 Principles of Isoelectric Focusing
163(1)
4.7.5.2 The Process of Isoelectric Focusing and the Estimation of pI
164(1)
4.7.5.3 Establishing a pH Gradient
164(1)
4.7.6 Two-dimensional Gel Electrophoresis
165(1)
4.7.6.1 Sample Preparation
166(1)
4.7.6.2 Protocol for SDS-PAGE
166(1)
4.7.7 Capillary Electrophoresis
166(3)
5 Molecular Biology 169(72)
5.1 Introduction
169(1)
5.2 Structure of Nucleic Acids
170(11)
5.2.1 The Nature and Components of Nucleic Acids
170(3)
5.2.2 Primary Structure of Nucleic Acids
173(1)
5.2.3 Secondary and Tertiary Structures of Nucleic Acids
174(5)
5.2.3.1 A Model for DNA Structure: The Double Helix
174(4)
5.2.3.2 Structures of Single-Stranded Nucleic Acids (RNA)
178(1)
5.2.3.3 Super Coiled Tertiary Structures of Closed DNA
178(1)
5.2.4 Stability of Secondary and Tertiary Structures of Nucleic Acids
179(1)
5.2.5 Physical Organization of DNA within the Nucleus
180(1)
5.3 Functions of Nucleic Acids
181(6)
5.3.1 Replication: DNA to DNA
182(1)
5.3.2 Transcription: DNA to RNA
183(2)
5.3.3 Translation: RNA to Protein
185(2)
5.4 Genes and Genome
187(3)
5.4.1 Introduction
187(1)
5.4.2 Genome Complexity
188(2)
5.5 Isolation and Separation of Nucleic Acids
190(5)
5.5.1 Conventional Chemical Nucleic Acid Extraction Methods
191(1)
5.5.2 Solid Phase Nucleic Acid Extraction using Silica-based Technology
192(1)
5.5.3 Magnetic Beads-based Nucleic Acid Isolation
192(1)
5.5.4 Anion Exchange Technology
193(1)
5.5.5 Automated Extraction Systems
193(1)
5.5.6 Electrophoresis for Separation of Nucleic Acids
193(2)
5.6 Manipulation and Detection of Nucleic Acids
195(12)
5.6.1 Enzymes Used to Manipulate Nucleic Acids in Molecular Biology
195(3)
5.6.1.1 Nucleases
195(2)
5.6.1.2 Ligases
197(1)
5.6.1.3 Polymerases
197(1)
5.6.1.4 DNA Modifying Enzymes
197(1)
5.6.2 Nucleic Acid Mutagenesis
198(4)
5.6.2.1 Oligonucleotide-directed Mutagenesis
199(1)
5.6.2.2 PCR-based Site-directed Mutagenesis
199(2)
5.6.2.3 CRISPR/Cas-9 Technology
201(1)
5.6.3 Nucleic Acid Hybridization
202(5)
5.6.3.1 Blotting
204(1)
5.6.3.2 Colony Hybridization
205(1)
5.6.3.3 Fluorescence in situ Hybridization
206(1)
5.7 Polymerase Chain Reaction
207(9)
5.7.1 Introduction
207(1)
5.7.2 Steps in PCR
208(2)
5.7.3 Variations of PCR
210(6)
5.7.3.1 Nested PCR
210(1)
5.7.3.2 Quantitative PCR (qPCR)
210(4)
5.7.3.3 Reverse Transcription PCR (RT-PCR)
214(1)
5.7.3.4 Inverse PCR
214(1)
5.7.3.5 Multiplex PCR
215(1)
5.7.3.6 Touchdown PCR
216(1)
5.7.3.7 Asymmetric PCR
216(1)
5.8 Nucleic Acid Sequencing
216(10)
5.8.1 First Generation Sequencing
217(1)
5.8.1.1 Chain Termination/Sanger Sequencing Method
217(1)
5.8.1.2 Maxim and Gilbert Sequencing
217(1)
5.8.1.3 Automated Fluorescent DNA Sequencing
218(1)
5.8.2 Next Generation (Second Generation) Sequencing
218(5)
5.8.2.1 Pyrosequencing
219(1)
5.8.2.2 Illumina
220(1)
5.8.2.3 Ion Torrent Sequencing
221(1)
5.8.2.4 Solid
222(1)
5.8.3 Third Generation Sequencing
223(3)
5.8.3.1 SMRT
224(1)
5.8.3.2 Oxford Nanopore
224(2)
5.9 Analyzing Gene and Gene Expression
226(10)
5.9.1 Methods for the Study of Gene Expression
227(9)
5.9.1.1 Low-Throughput Methods
227(6)
5.9.1.2 High-Throughput Methods
233(3)
5.10 Protocols
236(5)
5.10.1 Agarose Gel Electrophoresis
236(1)
5.10.2 Extraction of RNA Using the PureLink RNA Mini Kit
237(1)
5.10.3 Polymerase Chain Reaction (PCR)
238(3)
6 Cell Culture 241(41)
6.1 Introduction
241(1)
6.2 Cell Structure and Function
242(4)
6.3 Cell Cycle
246(5)
6.3.1 Mitosis
248(3)
6.4 Cell Microenvironment
251(1)
6.5 Primary Explantation versus Disaggregation
252(3)
6.6 Proliferation versus Differentiation
255(2)
6.6.1 Proliferation
255(1)
6.6.2 Differentiation
256(1)
6.7 Organotypic Culture
257(1)
6.8 Basics of Cell Culturing and Associated Measurements
258(7)
6.8.1 Types of Cell Culture
260(5)
6.8.1.1 Primary Cell Cultures
260(1)
6.8.1.2 Secondary Cell Culture
261(1)
6.8.1.3 Cell Line
261(1)
6.8.1.4 Stem Cell Cultures
261(1)
6.8.1.5 Subcultures
262(2)
6.8.1.6 Growth Cycle
264(1)
6.8.1.7 Serial Subculture
265(1)
6.9 Propagation, Population Doubling and Passage Number
265(1)
6.10 Cell Viability
265(1)
6.10.1 Common Assays for Cell Viability
265(1)
6.11 Cryopreservation
266(1)
6.12 Characterization and Validation
267(1)
6.12.1 Characterization
267(1)
6.12.2 Cross Contamination
267(1)
6.12.3 Microbial Contamination
268(1)
6.13 Microscopy
268(14)
6.13.1 The Light Microscope
269(1)
6.13.2 Fluorescence Microscopy
270(1)
6.13.3 Confocal Microscopy
271(1)
6.13.4 Electron Microscope
272(2)
6.13.5 Atomic Force Microscopy
274(8)
7 Antibody Technology 282(36)
7.1 Introduction to Immunochemical Techniques
282(1)
7.2 Antibodies
283(3)
7.3 Epitope Mapping
286(1)
7.4 Immunoassay
287(1)
7.4.1 Heterogenous Immunoassays Can Be Competitive or Noncompetitive
288(1)
7.5 ELISA
288(6)
7.5.1 Direct ELISA
289(1)
7.5.2 Indirect ELISA
290(1)
7.5.3 Sandwich ELISA
291(1)
7.5.4 Competitive/Inhibition ELISA
292(2)
7.6 Immunofluorescence
294(2)
7.7 Immunoblotting
296(1)
7.8 Immunoprecipitation Reaction
297(5)
7.8.1 How does IP work?
297(1)
7.8.2 Types of Immunoprecipitation (IP)
298(4)
7.9 Immunodiffusion
302(2)
7.9.1 Radial Immunodiffusion (RID)
302(1)
7.9.2 Ouchterlony Double Immunodiffusion (ODI)
303(1)
7.10 Radioimmunoassay (RIA)
304(2)
7.11 Immunoelectrophoresis
306(2)
7.12 Immunosensors
308(1)
7.12.1 Surface Plasmon Resonance (SPR)
309(1)
7.13 Immunotherapy
309(9)
7.13.1 General Principles of mAb Activity
312(1)
7.13.2 Targets of Therapeutic Antibodies
313(1)
7.13.3 Modifications
314(4)
Appendices 318(19)
Appendix 1 Troubleshooting: Cell Culture
318(4)
Appendix 2 Laboratory Safety
322(2)
Appendix 3 Statistics
324(6)
Appendix 4 Significant Figures
330(2)
Appendix 5 Units in the Biochemistry Laboratory
332(2)
Appendix 6
Chapter Contributors
334(1)
Appendix 7 Online Resources
334(3)
Index 337(4)
Color Plates 341
Bal Ram Singh is currently working as Director, Institute of Advanced Science, Dartmouth, USA. He had been a principal investigator at National Institutes of Health (NIH) and Department of Defense, USA. He has been on Blue-Ribbon Panels for NIH on the Biodefense Research, and has served on several NIH study section reviewer panels. He has published more than 180 papers in peer-reviewed journals. He had taught courses including biophysical techniques, biological spectroscopy, fluorescence spectroscopy and chemical biology at graduate level. He is a member of American Chemical Society, American Society for Microbiology, American Association for the Advancement of Science and American Society for Biochemistry and Molecular Biology. Raj Kumar is an Assistant Professor, Institute of Advanced Science, Dartmouth, USA. He did his Ph.D. from University of Massachusetts, Lowell. His areas of research include nanoparticles, biotechnology, drug delivery, cell culture and drug screening. He has published several papers in journals of national and international repute.