Human Genetic Disease Analysis

Part 1 Foetal DNA analysis, J.M. Old: chorionic villi DNA - sampling and shipment, DNA preparation, DNA yield; amniocyte DNA - choice of sample, DNA preparation; other DNA sources; quick DNA preparation for PCR; storage of DNA; DNA analysis - southern blot analysis, polymerase chain reaction; potential problems - maternal DNA contamination, plasmid and target DNA contamination, technical difficulties, non-paternity. Part 2 The use of synthetic oligonucleotides as specific hybridization probes in the diagnosis of genetic disorders, S.W. Thein, et al: principle of oligonucleotide hybridization; oligonucleotide probe design; detection of target DNA sequence - preparation of oligonucleotide probe, oligonucleotide probe hybridization, an alternative hybridization - competition hybridization; practical applications - polymorphism in the "c-erb-B2" transmembrane region, specificity of competition hybridization. Part 3 Pulsed-field gel electrophoresis in the analysis of genomic DNA and YAC clones, J.T. den Dunnen et al: available PFGE systems; preparation of DNA samples - isolation of DNA in agarose, preparation of marker DNAs, restriction digestion of agarose-embedded DNA; electrophoresis - the PFGE system, gel preparation, planned DNA separations, blotting of PFGE gels, hybridization of PFGE gels, rehybridization of PFGE blots; PFGE applications - analysis of chromosomal (human) DNA, YAC technology, further applications; future developments. Part 4 Fluorescent "in situ" hybridization, V.J. Buckle and K.A. Rack: practical considerations - chromosome and nuclei preparation, pre-hybridization procedures, DNA resources for use as probes, probe labelling, hybridization, signal detection, chromosome banding, visualization of signal, analysis of signal, trouble-shooting; applications - direct localization within the genome, sequence order on the chromosome, structural chromosome abnormalities, numerical chromosome abnormalities, chromosome painting, nuclear organization, other applications. Part 5 Fine mapping of genes - the characterization of the transcriptional unit, M. Antoniou, et al: cDNA and genomic DNA clones; mapping the transcriptional unit - restriction map, northern blots, "run-on" transcription, R looping, nuclease Sl protection and southern blots, nuclease Sl protection of end-labelled probes, exonuclease VII, T7, T3 or SP6 polymerase probes, primer extensions; polymerase chain reaction (PCR). Part 6 Chromosome analysis and sorting by flow cytometry, S. Monard et al: instrumentation - safety; discussion.