Gene Cloning and DNA Analysis

Terry Brown, University of Manchester, Faculty of Life Sciences, UK

PART 1 THE BASIC PRINCIPLES OF GENE CLONING AND DNA ANALYSIS.Chapter 1 Why Gene Cloning and DNA Analysis are Important.1.1 The early development of genetics.1.2 The advent of gene cloning and the polymerase chain reaction.1.3 What is gene cloning?.1.4 What is PCR?.1.5 Why gene cloning and PCR are so important.1.5.1 Gene isolation by cloning.1.5.2 Gene isolation by PCR.1.6 How to find your way through this book.Chapter 2 Vectors for Gene Cloning: Plasmids and Bacteriophages.2.1 Plasmids.2.1.1 Basic features of plasmids.2.1.2 Size and copy number.2.1.3 Conjugation and compatibility.2.1.4 Plasmid classification.2.1.5 Plasmids in organisms other than bacteria.2.2 Bacteriophages.2.2.1 Basic features of bacteriophages.2.2.2 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.2.2.3 Viruses as cloning vectors for other organisms.Chapter 3 Purification of DNA from Living Cells.3.1 Preparation of total cell DNA.3.1.1 Growing and harvesting a bacterial culture.3.1.2 Preparation of a cell extract.3.1.3 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.3.1.4 Concentration of DNA samples.3.1.5 Measurement of DNA concentration.3.1.6 Other methods for the preparation of total cell DNA.3.2 Preparation of plasmid DNA.3.2.1 Separation on the basis of size.3.2.2 Separation on the basis of conformation.Alkaline denaturation.Ethidium bromide-caesium chloride density gradient centrifugation.3.2.3 Plasmid amplification.3.3 Preparation of bacteriophage DNA.3.3.1 Growth of cultures to obtain a high l titre.3.3.2 Preparation of non-lysogenic l phages.3.3.3 Collection of phages from an infected culture.3.3.4 Purification of DNA from l phage particles.3.3.5 Purification of M13 DNA causes few problems.Chapter 4 Manipulation of Purified DNA.4.1 The range of DNA manipulative enzymes.4.1.1 Nucleases.4.1.2 Ligases.4.1.3 Polymerases.4.1.4 DNA modifying enzymes.4.1.5 Topoisomerases.4.2 Enzymes for cutting DNA - restriction endonucleases.4.2.1 The discovery and function of restriction endonucleases.4.2.2 Type II restriction endonucleases cut DNA at specific nucleotide sequences.4.2.3 Blunt ends and sticky ends.4.2.4 The frequency of recognition sequences in a DNA molecule.4.2.5 Performing a restriction digest in the laboratory.4.2.6 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.4.2.7 Estimation of the sizes of DNA molecules.4.2.8 Mapping the positions of different restriction sites in a DNA molecule.4.3 Ligation - joining DNA molecules together.4.3.1 The mode of action of DNA ligase.4.3.2 Sticky ends increase the efficiency of ligation.4.3.3 Putting sticky ends onto a blunt-ended molecule.Linkers.Adaptors.Chapter 5 Introduction of DNA into Living Cells.5.1 Transformation - the uptake of DNA by bacterial cells.5.1.1 Not all species of bacteria are equally efficient at DNA uptake.5.1.2 Preparation of competent E. coli cells.5.1.3 Selection for transformed cells.5.2 Identification of recombinants.5.2.1 Recombinant selection with pBR322 - insertional inactivation of an antibiotic.resistance gene.5.2.2 Insertional inactivation does not always involve antibiotic resistance.5.3 Introduction of phage DNA into bacterial cells.5.3.1 Transfection.5.3.2 In vitro packaging of l cloning vectors.5.3.3 Phage infection is visualized as plaques on an agar medium.5.4 Identification of recombinant phages.5.4.1 Insertional inactivation of a lacZ- gene carried by the phage vector.5.4.2 Insertional inactivation of the l cI gene.5.4.3 Selection using the Spi phenotype.5.4.4 Selection on the basis of l genome size.5.5 Introduction of DNA into non-bacterial cells.5.5.1 Transformation of individual cells.5.5.2 Transformation of whole organisms.Producing sticky ends by homopolymer tailing.Chapter 6 Cloning Vectors for E. coli.6.1 Cloning vectors based on E. coli plasmids.6.1.1 The nomenclature of plasmid cloning vectors.6.1.2 The useful properties of pBR322.6.1.3 The pedigree of pBR322.6.1.4 More sophisticated E. coli plasmid cloning vectors.pUC8 - a Lac selection plasmid.pGEM3Z - in vitro transcription of cloned DNA.6.2 Cloning vectors based on M13 bacteriophage.6.2.1 Development of the M13 cloning vectors.M13mp7 - symmetrical cloning sites.More complex M13 vectors.6.2.2 Hybrid plasmid-M13 vectors.6.3 Cloning vectors based on l bacteriophage.6.3.1 Segments of the l genome can be deleted without impairing viability.6.3.2 Natural selection can be used to isolate modified l that lack certain restriction.sites.6.3.3 Insertion and replacement vectors.Insertion vectors.Replacement vectors.6.3.4 Cloning experiments with l insertion or replacement vectors.6.3.5 Long DNA fragments can be cloned using a cosmid.6.4 l and other high capacity vectors enable genomic libraries to be constructed.6.5 Vectors for other bacteria...Chapter 7 Cloning Vectors for Eukaryotes.7.1 Vectors for yeast and other fungi.7.1.1 Selectable markers for the 2 m plasmid.7.1.2 Vectors based on the 2 m plasmid - yeast episomal plasmids.7.1.3 A YEp may insert into yeast chromosomal DNA.7.1.4 Other types of yeast cloning vector.7.1.5 Artificial chromosomes can be used to clone long pieces of DNA in yeast.The structure and use of a YAC vector.Applications for YAC vectors.7.1.6 Vectors for other yeasts and fungi.7.2 Cloning vectors for higher plants.7.2.1 Agrobacterium tumefaciens - nature's smallest genetic engineer.Using the Ti plasmid to introduce new genes into a plant cell.Production of transformed plants with the Ti plasmid.The Ri plasmid.Limitations of cloning with Agrobacterium plasmids.7.2.2 Cloning genes in plants by direct gene transfer.Direct gene transfer into the nucleus.Transfer of genes into the chloroplast genome.7.2.3 Attempts to use plant viruses as cloning vectors.Caulimovirus vectors.Geminivirus vectors.7.3 Cloning vectors for animals.7.3.1 Cloning vectors for insects.P elements as cloning vectors for Drosophila.Cloning vectors based on insect viruses.7.3.2 Cloning in mammals.Cloning vectors for mammals.Gene cloning without a vector.Chapter 8 How to Obtain a Clone of a Specific Gene.8.1 The problem of selection.8.1.1 There are two basic strategies for obtaining the clone you want.8.2 Direct selection.8.2.1 Marker rescue extends the scope of direct selection.8.2.2 The scope and limitations of marker rescue.8.3 Identification of a clone from a gene library.8.3.1 Gene libraries.8.3.2 Not all genes are expressed at the same time.8.3.3 mRNA can be cloned as complementary DNA.8.4 Methods for clone identification.8.4.1 Complementary nucleic acid strands hybridize to each other.8.4.2 Colony and plaque hybridization probing.8.4.3 Examples of the practical use of hybridization probing.Abundancy probing to analyse a cDNA library.Oligonucleotide probes for genes whose translation products have been.characterized.Heterologous probing allows related genes to be identified.8.4.4 Identification methods based on detection of the translation product of the.cloned gene.Antibodies are required for immunological detection methods.Using a purified antibody to detect protein in recombinant colonies.The problem of gene expression.Chapter 9 The Polymerase Chain Reaction.9.1 The polymerase chain reaction in outline.9.2 PCR in more detail.9.2.1 Designing the oligonucleotide primers for a PCR.9.2.2 Working out the correct temperatures to use.9.2.3 After the PCR: studying PCR products.Gel electrophoresis of PCR products.Cloning PCR products.9.3 Problems with the error rate of Taq polymerase.PART 2 THE APPLICATIONS OF GENE CLONING AND DNA ANALYSIS IN RESEARCH.Chapter 10 Studying Gene Location and Structure.10.1 How to study the location of a gene.10.1.1 Locating the position of a gene on a small DNA molecule.10.1.2 Locating the position of a gene on a large DNA molecule.Separating chromosomes by gel electrophoresis.In situ hybridization to visualize the position of a gene on a eukaryotic.chromosome.10.2 DNA sequencing - working out the structure of a gene.10.2.1 The Sanger-Coulson method - chain-terminating nucleotides.The primer.Synthesis of the complementary strand.Four separate reactions result in four families of terminated strands.Reading the DNA sequence from the autoradiograph.Not all DNA polymerases can be used for sequencing.10.2.2 Automated DNA sequencing.10.2.3 Sequencing PCR products.10.2.4 The Maxam-Gilbert method - chemical degradation of DNA.10.2.5 Building up a long DNA sequence.10.2.6 The achievements of DNA sequencing.Chapter 11 Studying Gene Expression and Function.11.1 Studying the transcript of a cloned gene.11.1.1 Electron microscopy of nucleic acid molecules.11.1.2 Analysis of DNA-RNA hybrids by nuclease treatment.11.1.3 Transcript analysis by primer extension.11.1.4 Other techniques for studying RNA transcripts.Northern hybridization.Reverse transcription-PCR (RT-PCR).Rapid amplification of cDNA ends (RACE).RNA sequencing.11.2 Studying the regulation of gene expression.11.2.1 Identifying protein binding sites on a DNA molecule.Gel retardation of DNA-protein complexes.Footprinting with DNase I.Modification interference assays.11.2.2 Identifying control sequences by deletion analysis.Reporter genes.Carrying out a deletion analysis.11.3 Identifying and studying the translation product of a cloned gene.11.3.1 HRT and HART can identify the translation product of a cloned gene.11.3.2 Analysis of proteins by in vitro mutagenesis.Different types of in vitro mutagenesis techniques.Using an oligonucleotide to create a point mutation in a cloned gene.Other methods of creating a point mutation in a cloned gene.The potential of in vitro mutagenesis.11.3.3 Studying protein-protein interactions.Phage display.The yeast two hybrid system.Chapter 12 Studying Genomes.12.1 Genomics - how to sequence a genome.12.1.1 The shotgun approach to genome sequencing.The H. influenzae genome sequencing project.Problems with shotgun cloning.12.1.2 The clone contig approach.Clone contig assembly by chromosome walking.Rapid methods for clone contig assembly.Clone contig assembly by sequence tagged site content analysis.12.1.3 Using a map to aid sequence assembly.Genetic maps.Physical maps.The importance of a map in sequence assembly.12.2 Post-genomics - trying to understand a genome sequence.12.2.1 Identifying the genes in a genome sequence.Searching for open reading frames.Distinguishing real genes from chance ORFs.12.2.2 Determining the function of an unknown gene.12.3 Studies of the transcriptome and proteome.12.3.1 Studying the transcriptome.12.3.2 Studying the proteome.PART 3 THE APPLICATIONS OF GENE CLONING AND DNA ANALYSIS IN BIOTECHNOLOGY.Chapter 13 Production of Protein from Cloned Genes.13.1 Special vectors for expression of foreign genes in E. coli.13.1.1 The promoter is the critical component of an expression vector.The promoter must be chosen with care.Examples of promoters used in expression vectors.13.1.2 Cassettes and gene fusions.13.2 General problems with the production of recombinant protein in E. coli.13.2.1 Problems resulting from the sequence of the foreign gene.13.2.2 Problems caused by E. coli.13.3 Production of recombinant protein by eukaryotic cells.13.3.1 Recombinant protein from yeast and filamentous fungi.Saccharomyces cerevisiae as the host for recombinant protein synthesis.Other yeasts and fungi.13.3.2 Using animal cells for recombinant protein production.Protein production in mammalian cells.Protein production in insect cells.13.3.3 Pharming - recombinant protein from live animals and plants.Pharming in animals.Recombinant proteins from plants.Ethical concerns raised by pharming.Chapter 14 Gene Cloning and DNA Analysis in Medicine.14.1 Production of recombinant pharmaceuticals.14.1.1 Recombinant insulin.Synthesis and expression of artificial insulin genes.14.1.2 Synthesis of human growth hormones in E. coli.14.1.3 Recombinant factor VIII.14.1.4 Synthesis of other recombinant human proteins.14.1.5 Recombinant vaccines.Producing vaccines as recombinant proteins.Recombinant vaccines in transgenic plants.Live recombinant virus vaccines.14.2 Identification of genes responsible for human diseases.14.2.1 How to identify a gene for a genetic disease.Locating the approximate position of the gene in the human genome.Identification of candidates for the disease gene.14.3 Gene therapy.14.3.1 Gene therapy for inherited diseases.14.3.2 Gene therapy and cancer.14.3.3 The ethical issues raised by gene therapy..Chapter 15 Gene Cloning and DNA Analysis in Agriculture.15.1 The gene addition approach to plant genetic engineering. 15.1.1 Plants that make their own insecticides. The d-endotoxins of Bacillus thuringiensis. Cloning a d-endotoxin gene in maize. Cloning d-endotoxin genes in chloroplasts. Countering insect resistance to d-endotoxin crops. 15.1.2 Herbicide resistant crops. 'Roundup Ready' crops. A new generation of glyphosate resistant crops. 15.1.2 Other gene addition projects.15.2 Gene subtraction. 15.2.1 The principle behind antisense technology. 15.2.2 Antisense RNA and the engineering of fruit ripening in tomato. Using antisense RNA to inactivate the polygalacturonase gene. Using antisense RNA to inactivate ethylene synthesis. 15.2.3 Other examples of the use of antisense RNA in plant genetic engineering.15.3 Problems with genetically modified plants. 15.3.1 Safety concerns with selectable markers. 15.3.2 The terminator technology. 15.3.3 The possibility of harmful effects on the environment.Chapter 16 Gene Cloning and DNA Analysis in Forensic Science and Archaeology.16.1 DNA analysis in the identification of crime suspects. 16.1.1 Genetic fingerprinting by hybridization probing. 16.1.2 DNA profiling by PCR of short tandem repeats.16.2 Studying kinship by DNA profiling. 16.2.1 Related individuals have similar DNA profiles. 16.2.2 DNA profiling and the remains of the Romanovs. STR analysis of the Romanov bones. The missing children.16.3 Sex identification by DNA analysis. 16.3.1 PCRs directed at Y chromosome-specific sequences. 16.3.2 PCR of the amelogenin gene.16.4 Archaeogenetics - using DNA to study human evolution. 16.4.1 The origins of modern humans. DNA analysis has challenged the multiregional hypothesis. DNA analysis shows that Neanderthals are not the ancestors of modern. Europeans. 16.4.2 DNA can also be used to study prehistoric human migrations. The spread of agriculture into Europe. Using mitochondrial DNA to study past human migrations into Europe