Advanced Molecular Genetics

The development of powerful new techniques and refmements of tech niques in molecular genetics in recent years, and the surge in interest in biotechnology based on genetic methods, have heralded a new golden age in molecular genetics, and stimulated in diverse disciplines much interest in the technologies themselves and their potential uses in basic and applied biomedical sciences. Although some excellent specialist laboratory manuals (especially the Cold Spring Harbor Laboratory manuals by I. H. Miller; R. W. Davies et al. ; and T. Maniatis et al. ) on certain chapters of molecular genetics exist, no general text that covers a broad spectrum of the sub ject has thus far been published. The purpose of this manual is to pre sent most, though of necessity not all of the important methods of molecular genetics, in a series of simple experiments, many of which can be readily accomplished by the microbiologist, biochemist or biotechnologist that has had only limited exposure to genetics. The remainder of the experiments require either greater familiarity with the subject, or guidance by someone with such experience. The book should, therefore, not only enable individuals to acquire new proce dures for ongoing projects, but also serve as a basis for the teaching of molecular genetic techniques in formal predoctoral and postdoctoral laboratory courses.

1 Basic Methods.- 1.1 Genetic Transfer in Prokaryotes: Transformation, Transduction, and Conjugation.- 1.2 Isolation of Plasmid DNA.- 1.3 Characterization of Plasmid DNA by Agarose Gel Electrophoresis.- 1.4 Characterization of Plasmid in Wild Strains of Bacteria.- 1.5 Mobilization of Host Chromosomal Genes and Vector Plasmids in E. coli by the P-Type Plasmid R68.45.- 1.6 Characterization of Plasmid Relaxation Complexes.- 1.7 Use of the BAL31 Exonuclease to Map Restriction Endonucleases Cleavage Sites in Circular Genomes.- 1.8 IncPl Typing of Plasmid DNA by Southern Hybridization.- 1.9 Molecular Epidemiology by Colony Hybridization Using Cloned Genes.- 2 Mutagenesis.- 2.1 Isolation of Suppressible Mutations by Hydroxylamine Mutagenesis in Vitro.- 2.2 Transposition of Tni to the Phage PI Genome: Isolation of Restriction Deficient Mutants.- 2.3 In Vivo Genetic Engineering: Use of Transposable Elements in Plasmid Manipulation and Mutagenesis of Bacteria Other Than E. coli.- 2.4 Generation of Deletion Mutations in Vitro with the BAL31 Exonuclease.- 3 Gene Cloning.- 3.1 Gene Cloning: An Introduction.- 3.2 Ligation of Cohesive-Ended and Flush-Ended DNA Fragments.- 3.3 Cloning with Plasmid Vectors.- 3.4 Mini-Plasmid Formation: The Cloning of Replication Origins.- 3.5 Gene Cloning with Bacteriophage ?.- 3.6 Cloning with Cosmids.- 4 Gene Expression.- 4.1 Synthesis of Plasmid-Encoded Polypeptides in Maxicells.- 4.2 Synthesis of Bacteriophage and Plasmid-Eneoded Polypeptides in Minicells.- 4.3 Determination of Coding Regions on Multicopy Plasmids: Analysis of the Chloramphenicol Acetyltransferase Gene of Plasmid pACYC184.- 4.4 Identification of Gene Products by Coupled Transcription- Translation of DNA Fragments in Cell-Free Extracts of E. coli.- 5 DNA Sequencing.- 6 Electron Microscopy.- 7 Transcription.- 7.1 Mapping of RNA Polymerase Binding Sites by Electron Microscopy.- 7.2 Determination of the Startpoints and Orientation of in Vitro Transcripts by Electron Microscopy of R-Loops.- 8 DNA Replication.- 8.1 Localization of Origins of Plasmid DNA Replication.- 8.2 Plasmid DNA Replication in Vitro.