Extraordinary advances have been made in the field of human molecular genetics during the past five years. The ability to amplify a specific region ofDNA a millionfold in a few hours using the polymerase chain reaction has led to the rapid identification of mutations in human disease and of DNA sequence polymorphisms on every human chro- some. DNA fragments of up to 1 megabase in length can now be resolved by pulsed-field gel electrophoresis to create long-range physical maps of important regions of the genome, and can be cloned in the form of yeast artificial chromosomes. The discovery of highly variable "minisatellite" DNA sequences has led to the development of DNA fingerprinting. The application of these techniques to the study of the human genome has culminated in major advances such as the cloning of the cystic fibrosis gene, the construction of genetic linkage maps of each human chro- some, the mapping of many genes responsible for human inherited d- orders, genetic fingerprinting of forensic specimens, and the detection of mutations involved in the development of human tumors. Although many of the new techniques in molecular genetics can be learned relatively easily, it is often difficult for a researcher to obtain all of the relevant information necessary for getting up a technique and applying it successfully. The information available in the research lite- ture often lacks the depth of description that the new user requires.
Table of ContentsThe Polymerase Chain Reaction: Getting Started. Direct DNA Sequencing of Complementary DNA Amplified by the Polymerase Chain Reaction. Direct-Sequencing of PCR-Amplified DNA. Rapid DNA Sequence Analysis Using Fluorescent Labels. Detection of Mutations in DNA and RNA by Chemical Cleavage. Rapid Methods for Detection of Polymorphic Markers in Genomic DNA. The Analysis of Point Mutations Using Synthetic Oligonucleotide Probes. Detection of Mutations by the Amplification Refractory Mutation System (ARMS). Automated Gene Detection Using the Oligonucleotide Ligation Assay. Detection of Point Mutations by Denaturing-Gradient Gel Electrophoresis. The Detection and Mapping of Point Mutations by RNase A Cleavage. Discontinuous Polyacrylamide Gel Electrophoresis of DNA Fragments. Extraction and Enzymatic Amplification of DNA from Paraffin-Embedded Specimens. The Use of the Polymerase Chain Reaction in the Mapping of Human Genes Using Somatic Cell Hybrids. The Southern Blot: An Update. The Detection of Specific DNA Sequences by Enhanced Chemiluminescence. Pulsed-Field Gel Electrophoresis. Cloning from Gels Following Pulse-Field Gel Electrophoresis. Yeast Artificial-Chromosome (YAC) Cloning Systems. Gene Targeting for Somatic Cell Manipulation. In Situ Hybridization of Chromosomes. DNA Fingerprinting Analysis: Methodology and Its Applications. DNA Fingerprinting and Forensic Medicine. The Detection of Point Mutations in Hemoglobin Defects Using Allele-Specific Oligonucleotide Probes. Detection of Gene Deletions Using Multiplex Polymerase Chain Reactions. Application of Pulsed-Field Gel Electrophoresis to Genetic Diagnosis. Molecular Diagnostics of Cancer. The Detection of Latent Virus Infection by Polymerase Chain Reaction. Mapping Inherited Diseases by Linkage Analysis. Diagnosis of Genetic Disorders with Linked DNA Markers. Software for Genetic Linkage Analysis. Creating Animal Models of Genetic Diseases. Molecular Biology and Medicine: Ethical Implications. Appendix. Index.