BIOL 311 Human Genetics Fall 2006Lecture: GenomicsReading: Chap. 8 pp. 210-221Outline:1. The path to the genome project2. Human genetic map3. Mapping genes and markers to chromosomes4. Physical mapping5. Sequencing strategiesLecture:1. The path to the human genome projectOne of the most important discoveries of science in this century is the deduction of the entire base sequence of the human genome. The draft sequence was completed in June 2000 and now the sequence of the entire genome is essentially complete. Milestones: Box 8.31956 Human cells shown to contain 46 chromosomes1977 Sanger developed dideoxy sequencing1981 Sander published complete sequence of human mitochondrial DNA1990 Human genome project launched as $3 billion, 15 year project1992 French group published first human genetic linkage map1995 Lander et al. published first detailed physical map of the human genome1999 First human chomosome, #22, completely sequenced2000 Draft of human genome announced by Celer and International Human Gene Sequencing Consortium2001 Papers in Science and Nature on draft sequence2003 Human genome project completed2. Human genetic mapa. Gene mapping1- only a few hundred genes known in early days of genome project- genes vary in length, location- therefore need more sites for detailed mapb. RFLP markers- segments of DNA that vary in sites for restriction enzymes- can be far apart- not easy to typec. microsatellite markers (SSR) simple sequence repeats- informative, easy to type- dispersed throughout the genome- example: CACACA…o GTGTGT…- Also used in forensic analysis3. Mapping genes and markers to chromosomesa. FISH: fluorescence in situ hybridization- Fluorescently tagged DNA probe- Incubate with lysed cells with denatured chromosomes on a slideb. Somatic Cell Hybrids Box 8.4[locate image]Fuse human cell with mouse or hamster cell in presence of PEG to form heterokaryonrandom loss of most human chromosomes, while all rodent chromosomes are keptcreate hybrid panel--different cell lines retaining particular human chromosomes or parts of chromosomesTest for presence of protein or enzyme produced by particular gene and presence of a particular human chromosomeORCarry out a Southern blot of DNA from a panel probed with a particular cDNA or gene probe.c. Radiation HybridsMap genes to particular segment of a chromosome[locate image]Human cells are exposed to a lethal dose of radiationRadiation induces chromosome breaksIrradiated human cells are fused with rodent cellsGenetic selection (thymidine kinase +) with dominant selectable marker used to identify rodent cells that have retained some human chromosome fragmentsCompare pattern of hybridization of unmapped DNA sequence to that of known STS (sequence tagged sites--unique DNA sequences)24. Physical mapping Box 8.5- determine number of bases separating DNA markers- partial digest of genomic DNA cut occasionally at restriction sites- clone fragments into large capacity vector (YAC, BAC, PAC)- align overlapping clones to create a "contig"overlaps are based on restriction siteslocation of markers referred as STS=sequence tagged site5. Sequencing strategies for the genome projecta. International Human Genome ProjectUS Human Genome Project/Francis CollinsFig. 8.3 Used physical maps to align fragments b. CeleraUS Company/Craig Venter"shotgun"