Biotechnology (eBook)

Applying the Genetic Revolution

David P. Clark, Nanette J. Pazdernik (Autoren)

eBook Download: EPUB

2010 | 1. Auflage
768 Seiten
Elsevier Science (Verlag)
978-0-08-088793-7 (ISBN)

Infos

Unlike most Biotechnology textbooks, Dr. David P. Clark's Biotechnology approaches modern Biotechnology from a Molecular Basis, which grew out of the increasing biochemical understanding of physiology. Using straight forward, less-technical jargon, Clark manages to introduce each chapter with a basic concept, that ultimately evolves into a more specific detailed principle. This up-to-date text covers a wide realm of topics including forensics and bioethics using colorful illustrations and concise applications.

This book will help readers understand what molecular biotechnology actually is as a scientific discipline, how the research in this area is conducted, and how this technology may impact the future.

? Up-to-date text focuses on modern biotechnology with a molecular foundation
? Basic concepts followed by more detailed, specific applications
? Clear, color illustrations of key topics and concepts
? Clearly written without overly technical jargon or complicated examples

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Unlike most biotechnology textbooks, Dr. David P. Clark's Biotechnology approaches modern biotechnology from a molecular basis, which grew out of the increasing biochemical understanding of physiology. Using straightforward, less-technical jargon, Clark manages to introduce each chapter with a basic concept that ultimately evolves into a more specific detailed principle. This up-to-date text covers a wide realm of topics, including forensics and bioethics, using colorful illustrations and concise applications.This book will help readers understand molecular biotechnology as a scientific discipline, how the research in this area is conducted, and how this technology may impact the future.* Up-to-date text focuses on modern biotechnology with a molecular foundation* Basic concepts followed by more detailed, specific applications * Clear, color illustrations of key topics and concepts * Clearly written without overly technical jargon or complicated examples

Inhalt

Front Cover 1
Calculations in Molecular Biology and Biotechnology 4
Copyright Page 5
Contents 8
Foreword 15
Chapter 1. Scientific Notation and Metric Prefixes 18
Introduction 18
Significant Digits 18
Rounding Off Significant Digits in Calculations 19
Exponents and Scientific Notation 21
Expressing Numbers in Scientific Notation 21
Converting Numbers from Scientific Notation to Decimal Notation 23
Adding and Subtracting Numbers Written in Scientific Notation 25
Multiplying and Dividing Numbers Written in Scientific Notation 26
Metric Prefixes 30
Conversion Factors and Canceling Terms 31
Chapter 2. Solutions Mixtures and Media 35
Introduction 35
Calculating Dilutions: A General Approach 35
Concentrations by a Factor of X 37
Preparing Percent Solutions 39
Diluting Percent Solutions 40
Moles and Molecular Weight: Definitions 44
Molarity 45
Diluting Molar Solutions 48
Converting Molarity to Percent 49
Converting Percent to Molarity 50
Normality 51
PH 52
pKa and the Henderson–Hasselbalch Equation 56
Chapter 3. Cell Growth 59
The Bacterial Growth Curve 59
Manipulating Cell Concentration 63
Plotting OD550 vs. Time on a Linear Graph 65
Plotting the Logarithm of OD550 vs. Time on a Linear Graph 66
Plotting the Log of Cell Concentration vs. Time 68
Calculating Generation Time 69
Plotting Cell Growth Data on a Semilog Graph 72
Determining Generation Time Directly from a Semilog Plot of Cell Concentration vs. Time 76
Plotting Cell Density versus OD550 on a Semilog Graph 77
The Fluctuation Test 78
Fluctuation Test Example 80
Variance 81
Measuring Mutation Rate 83
Measuring Cell Concentration on a Hemocytometer 92
Chapter 4. Working with Bacteriophage 94
Introduction 94
Multiplicity of Infection 94
Probabilities and Multiplicity of Infection 96
Measuring Phage Titer 102
Diluting Bacteriophage 103
Measuring Burst Size 104
Chapter 5. Quantitation of Nucleic Acids 107
Quantitation of Nucleic Acids by Ultraviolet Spectroscopy 107
Determining the Concentration of Double-Stranded DNA 108
Using Absorbance and an Extinction Coefficient to Calculate Double-Stranded DNA Concentration 111
Calculating DNA Concentration as a Millimolar (mM) Amount 113
Determining the Concentration of Single-Stranded DNA Molecules 114
Oligonucleotide Quantitation 116
Measuring RNA Concentration 120
Molecular Weight, Molarity, and Nucleic Acid Length 121
Estimating DNA Concentration on an Ethidium Bromide–Stained Gel 125
Chapter 6. Labeling Nucleic Acids with Radioisotopes 126
Introduction 126
Using Radioactivity: The Curie 126
Estimating Plasmid Copy Number 127
Labeling DNA by Nick Translation 129
Random Primer Labeling of DNA 131
Labeling 3' Termini with Terminal Transferase 136
cDNA Synthesis 138
Homopolymeric Tailing 145
In Vitro Transcription 150
Chapter 7. Oligonucleotide Synthesis 153
Introduction 153
Synthesis Yield 153
Measuring Stepwise and Overall Yield by the DMT Cation Assay 156
Overall Yield 156
Stepwise Yield 157
Calculating Micromoles of Nucleoside Added at Each Base Addition Step 159
Chapter 8. The Polymerase Chain Reaction 160
Introduction 160
Template and Amplification 160
Exponential Amplification 162
PCR Efficiency 164
Calculating the Tm of the Target Sequence 168
Primers 170
Primer Tm 175
dNTPs 182
DNA Polymerase 185
Quantitative PCR 188
Chapter 9. Recombinant DNA 203
Introduction 203
Restriction Endonucleases 203
The Frequency of Restriction Endonuclease Cut Sites 205
Calculating the Amount of Fragment Ends 206
Ligation 209
Transformation Efficiency 224
Genomic Libraries: How Many Clones Do You Need? 225
cDNA Libraries: How Many Clones Are Enough? 227
Expression Libraries 228
Screening Recombinant Libraries by Hybridization to DNA Probes 229
Sizing DNA Fragments by Gel Electrophoresis 241
Generating Nested Deletions Using Nuclease BAL 31 254
Chapter 10. Protein 259
Introduction 259
Protein Quantitation by Measuring Absorbance at 280 nm 259
Using Absorbance Coefficients and Extinction Coefficients to Estimate Protein Concentration 260
Relating Absorbance Coefficient to Molar Extinction Coefficient 262
Determining a Protein's Extinction Coefficient 263
Relating Concentration in Milligrams per Milliliter to Molarity 265
Protein Quantitation Using A280 When Contaminating Nucleic Acids Are Present 266
Protein Quantitation at 205 nm 267
Protein Quantitation at 205 nm When Contaminating Nucleic Acids Are Present 268
Measuring Protein Concentration by Colorimetric Assay- The Bradford Assay 269
Using ß-Galactosidase to Monitor Promoter Activity and Gene Expression 271
Specific Activity 274
The CAT Assay 277
Use of Luciferase in a Reporter Assay 282
In Vitro Translation–Determining Amino Acid Incorporation 283
Chapter 11. Centrifugation 287
Introduction 287
Relative Centrifugal Force (g Force) 287
Converting g Force to Revolutions per Minute 289
Determining g Force and Revolutions per Minute by Use of a Nomogram 290
Calculating Sedimentation Times 292
Chapter 12. Forensic Science 295
Introduction 295
Alleles and Genotypes 295
Calculating Genotype Frequencies 297
Calculating Allele Frequencies 298
The Hardy–Weinberg Equation and Calculating Expected Genotype Frequencies 299
The Chi-Square Test: Comparing Observed to Expected Values 303
Sample Variance 307
Sample Standard Deviation 308
Pi: The Power of Inclusion 309
Pd: The Power of Discrimination 310
DNA Typing and a Weighted Average 311
The Multiplication Rule 312
Index 314

Textauszug

List of Figures

Chapter 1. Basics of Biotechnology

Figure 1.1. Traditional Biotechnology ProductsBread, cheese, wine, and beer have been made worldwide for many centuries using microorganisms, such as yeast.

Figure 1.2. Teosinte versus Modern cornThroughout history, people have improved many plants for higher yields. Teosinte (smaller cob and green seeds) is considered the ancestor of commercial corn (larger cob; a blue-seeded variety is shown). Courtesy of Wayne Campbell, Hila Science Camp.

Figure A. Relationship of Genotype and Phenotype(A) Each parent has two alleles, either two yellow or two green. Any offspring will be heterozygous, each having a yellow and a green allele. Since the yellow allele is dominant, the peas look yellow. (B) When the heterozygous F1 offspring self-fertilize, the green phenotype re-emerges in one-fourth of the F2 generation.

Figure 1.3. Nucleic Acid Structure(A) DNA has two strands antiparallel to each other. The structure of the subcomponents is shown to the sides.(B) RNA is usually single-stranded and has two chemical differences from DNA. First, an extra hydroxyl group (-OH) is found at the 2? position of ribose, and second, thymine is replaced by uracil.

Figure 1.4. Packaging of DNA in Bacteria and Eukaryotes(A) Bacterial DNA is supercoiled and attached to a scaffold to condense its size to fit inside the cell. (B) Eukaryotic DNA is wrapped around histones to form a nucleosome. Nucleosomes are further condensed into a 30-nm fiber attached to proteins at MAR sites.

Figure 1.5. Hydrothermal Vent TubewormsThese hydrothermal vent tubeworms from the Pacific Ocean get energy from symbiotic bacteria that live inside them. Courtesy of National Oceanic & Atmospheric Administration/National Undersea Research Program (NURP).

Figure 1.6. Subcellular Structure of Escherichia coli(A) Scanning electron micrograph of E. coli. The rod-shaped bacteria are approximately 0.6 microns by 1–2 microns. Courtesy of Rocky Mountain Laboratories, NIAID, NIH. (B) Gram-negative bacteria have three structural layers surrounding the cytoplasm. The outer membrane and cytoplasmic membrane are lipid bilayers, and the cell wall is made of peptidoglycan. Unlike eukaryotes, no membrane surrounds the chromosome, leaving the DNA readily accessible to the cytoplasm.

Figure 1.7. Bacteria are Easy to Grow(A) Bacteria growing in liquid culture. (B) Bacteria growing on agar. This photo shows a mixture of bacterial colonies from the blue/white method for screening plasmid insertions—see Chapter 3 and Fig. 3.15 for a full explanation. (C) Fast-growing bacteria can double in numbers in short periods. Here, the number of bacteria double after approximately 45 minutes and reach a density of 5 × 109 cells/mL in about 5 hours.

Figure 1.8. The E. coli chromosomeThe E. coli chromosome is divided into 100 map units, arbitrarily starting at the thrABC operon. Various genes and their locations are shown. The replication origin (oriC) and termination zone (terB and terC) are indicated.

Figure 1.9. Plasmids Encode the Genes for ColicinColE1 plasmids are extrachromosomal DNA elements that are maintained by bacteria for producing a toxin (cea gene). They also carry genes for toxin release and immunity. These plasmids have been modified to carry genes useful in genetic engineering.

Figure 1.10. Somatic versus Germline CellsDuring development, cells either become somatic cells, which form the body, or germline cells, which form either eggs or sperm. The germline cells are the only cells whose genes are passed on to future generations.

Figure 1.11. Somatic MutationsThe early embryo has the same genetic information in every cell. During division of a somatic cell, a mutation may occur that affects the organ or tissue it gives rise to. Because the mutation was isolated in a single precursor cell, other parts of the body and the germline cells will not contain the mutation. Consequently, the mutation will not be passed on to any offspring.

Figure 1.12. Structure of Yeast CellThis yeast cell, undergoing division, is starting to partition components into the bud. Eventually, the bud will grow in size and be released from the mother (lower oval), leaving a scar on the surface of the cell wall.

Figure 1.13. The 2-Micron Plasmid of YeastTwo different forms of the 2-micron plasmid are shown. The enzyme Flp recombinase recognizes the FRT sites and recombines them, thus flipping one half of the plasmid relative to the other half.

Figure 1.14. Alternating Haploid and Diploid Phases of YeastHaploid cells come in two different forms, a and a. These express mating pheromones, a factor and alpha (a) factor, which attract the two forms to each other. When the pheromones bind to receptors on the opposite cell type, the two haploid cells become competent to fuse into a diploid cell. Diploid cells sporulate under growth limiting conditions. Otherwise, the diploid cells form genetic clones by budding.

Figure 1.15. Caenorhabditis elegansCourtesy of Jill Bettinger, Virginia Commonwealth University, Richmond, VA.

Figure 1.16. Life Cycle ofCaenorhabditis elegansWhen the C. elegans sperm fuses with an egg, a small worm develops (L1). The larva goes through multiple stages until it reaches the sexually mature adult phase. C. elegans has six different chromosomes: five autosomes and one X chromosome. The worms are diploid, with two sets of chromosomes. When the embryo has two X chromosomes, it becomes a hermaphrodite. If the embryo has only one X, it becomes a male, but males make up only 0.05% of a normal population. The genome is 97 Mb and was completely sequenced in 1998. Approximately 27% of the genome is coding sequence with about 19,000 genes, more than 900 of which are RNA coding genes. The average gene contains five introns and is about 3000 base pairs long. Intronic DNA accounts for 26% of the total genome. The remaining 47% of the genome is intergenic and noncoding.

Figure 1.17. Life Cycle ofDrosophila melanogasterDrosophila fruit flies start as a tiny egg that develops into a worm (maggot). After a series of larval stages, the worm forms a pupa where the adult form develops.

Figure 1.18. Polytene ChromosomeFluorescent staining of polytene chromosome from Drosophila.

Figure 1.19. The Zebrafish, Danio rerioThis fish is used as a model vertebrate to study genetics, cell biology, and developmental biology.

Figure 1.20. Human HeLa Cells Grown in VitroHeLa cells were taken from the tumor of Henrietta Lacks, a woman suffering from cervical cancer, in the 1950s and have been cultured continuously ever since. (A) Viewed under phase contrast. (B) Viewed under differential interference contrast. Courtesy of Michael W. Davidson, Optical Microscopy Group, National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida.

Figure 1.21. Insect Cells in Culture(A) HvT1 cells from tobacco budworm testes are strongly attached to the surface of the dish. (B) TN368 cells from cabbage looper ovary are only loosely attached. Courtesy of Dwight E. Lynn, Insect Biocontrol Lab, USDA, Beltsville, MD.

Figure 1.22. Arabidopsis thalianaThe plant most used as a model for molecular biology research is A. thaliana, a member of the mustard family (Brassicaceae). Courtesy of Dr. Jeremy Burgess, Science Photo Library.

Figure 1.23. Virus Life CycleThe life cycle of a virus starts when the viral DNA or RNA enters the host cell. Once inside, the virus uses the host cell to manufacture more copies of the virus genome and to make the protein coats for assembly of virus particles. Once multiple copies of the virus have been assembled, the host cell bursts open, allowing the progeny to escape and find other hosts to invade.

Figure 1.24. Examples of Different VirusesViruses come in a variety of shapes and sizes that determine whether the entire virus or only its genome enters the host cells.

Figure 1.25. Retroviral Life CycleRetroviral genomes are made of positive RNA. Once the RNA enters the host, a DNA copy of the genome is made using reverse transcriptase. The original RNA strand is then degraded and replaced with DNA. Then the entire double-stranded DNA version of the retrovirus genome can integrate into the host genome.

Figure 1.26. Conjugation in E. coliBacteria with a transferable plasmid can make a sex pilus that attaches to a recipient cell. When the two cells touch, a conjugation...

Produkt-Details

• Erscheint lt. Verlag: 21.7.2010
• Sprache: englisch
• Themenwelt: Sachbuch/Ratgeber
• Themenwelt: Medizin / Pharmazie ► Allgemeines / Lexika
• Themenwelt: Naturwissenschaften ► Biologie ► Biochemie
• Themenwelt: Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie
• Themenwelt: Technik ► Umwelttechnik / Biotechnologie
• ISBN-10: 0-08-088793-7 / 0080887937
• ISBN-13: 978-0-08-088793-7 / 9780080887937