| |
| |
| The Basic Principles of Gene Cloning and Dna Analysis | |
| |
| |
| Why Gene Cloning and DNA Analysis are Important | |
| |
| |
| The early development of genetics | |
| |
| |
| The advent of gene cloning and the polymerase chain reaction | |
| |
| |
| What is gene cloning? | |
| |
| |
| What is PCR? | |
| |
| |
| Why gene cloning and PCR are so important | |
| |
| |
| Gene isolation by cloning | |
| |
| |
| Gene isolation by PCR | |
| |
| |
| How to find your way through this book | |
| |
| |
| Vectors for Gene Cloning: Plasmids and Bacteriophages | |
| |
| |
| Plasmids | |
| |
| |
| Basic features of plasmids | |
| |
| |
| Size and copy number | |
| |
| |
| Conjugation and compatibility | |
| |
| |
| Plasmid classification | |
| |
| |
| Plasmids in organisms other than bacteria | |
| |
| |
| Bacteriophages | |
| |
| |
| Basic features of bacteriophages | |
| |
| |
| Lysogenic phages | |
| |
| |
| Gene organization in l DNA molecule | |
| |
| |
| The linear and circular forms of l DNA M13 - a filamentous phage | |
| |
| |
| The attraction of M13 as a cloning vector | |
| |
| |
| Viruses as cloning vectors for other organisms | |
| |
| |
| Purification of DNA from Living Cells | |
| |
| |
| Preparation of total cell DNA | |
| |
| |
| Growing and harvesting a bacterial culture | |
| |
| |
| Preparation of a cell extract | |
| |
| |
| Purification of DNA from a cell extract | |
| |
| |
| Removing contaminants by organic extraction and enzyme digestion | |
| |
| |
| Using ion-exchange chromatography to purify DNA from a cell extract | |
| |
| |
| Concentration of DNA samples | |
| |
| |
| Measurement of DNA concentration | |
| |
| |
| Other methods for the preparation of total cell DNA | |
| |
| |
| Preparation of plasmid DNA | |
| |
| |
| Separation on the basis of size | |
| |
| |
| Separation on the basis of conformation | |
| |
| |
| Alkaline denaturation | |
| |
| |
| Ethidium bromide-caesium chloride density gradient centrifugation | |
| |
| |
| Plasmid amplification | |
| |
| |
| Preparation of bacteriophage DNA | |
| |
| |
| Growth of cultures to obtain a high l titre | |
| |
| |
| Preparation of non-lysogenic l phages | |
| |
| |
| Collection of phages from an infected culture | |
| |
| |
| Purification of DNA from l phage particles | |
| |
| |
| Purification of M13 DNA causes few problems | |
| |
| |
| Manipulation of Purified DNA | |
| |
| |
| The range of DNA manipulative enzymes | |
| |
| |
| Nucleases | |
| |
| |
| Ligases | |
| |
| |
| Polymerases | |
| |
| |
| DNA modifying enzymes | |
| |
| |
| Topoisomerases | |
| |
| |
| Enzymes for cutting DNA - restriction endonucleases | |
| |
| |
| The discovery and function of restriction endonucleases | |
| |
| |
| Type II restriction endonucleases cut DNA at specific nucleotide sequences | |
| |
| |
| Blunt ends and sticky ends | |
| |
| |
| The frequency of recognition sequences in a DNA molecule | |
| |
| |
| Performing a restriction digest in the laboratory | |
| |
| |
| Analysing the result of restriction endonuclease cleavage | |
| |
| |
| Separation of molecules by gel electrophoresis | |
| |
| |
| Visualizing DNA molecules by staining a gel | |
| |
| |
| Visualizing DNA molecules by autoradiography | |
| |
| |
| Estimation of the sizes of DNA molecules | |
| |
| |
| Mapping the positions of different restriction sites in a DNA molecule | |
| |
| |
| Ligation - joining DNA molecules together | |
| |
| |
| The mode of action of DNA ligase | |
| |
| |
| Sticky ends increase the efficiency of ligation | |
| |
| |
| Putting sticky ends onto a blunt-ended molecule | |
| |
| |
| Linkers | |
| |
| |
| Adaptors | |
| |
| |
| Introduction of DNA into Living Cells | |
| |
| |
| Transformation - the uptake of DNA by bacterial cells | |
| |
| |
| Not all species of bacteria are equally efficient at DNA uptake | |
| |
| |
| Preparation of competent E. coli cells | |
| |
| |
| Selection for transformed cells | |
| |
| |
| Identification of recombinants | |
| |
| |
| Recombinant selection with pBR322 - insertional inactivation of an antibiotic.resistance gene | |
| |
| |
| Insertional inactivation does not always involve antibiotic resistance | |
| |
| |
| Introduction of phage DNA into bacterial cells | |
| |
| |
| Transfection | |
| |
| |
| In vitro packaging of l cloning vectors | |
| |
| |
| Phage infection is visualized as plaques on an agar medium | |
| |
| |
| Identification of recombinant phages | |
| |
| |
| Insertional | |