chapter 9-v2
TRANSCRIPT
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Chapter 9:DNA-Based Information Technologies
DNA cloning: the basics
Genes to genomes
Genomes to proteomes
Genome alterations/new biotechnology products
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Tuesday, Apr 24ESB 1001
3:30 PM (W.E. Palke Memorial Lecture)Bruce Berne- Columbia University
Hydrophobibity and Drying in Physical and Biological Systems
BMSE SeminarFriday April 27, 2007
3:00pmWebb 1100
SILVIA CAVAGNEROUniversity of Wisconsin-Madison
Department of ChemistryProtein folding at birth: experimental and computational
studies on and off the ribosome
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Biochemistry: isolation and study of individual cellular components and their reassembly
Much of this is defined by the genome
Ability to manipulate genes/genomes (1974): Berg/Boyer/Cohen
DNA cloning (recombinant DNA technology/genetic eng.)
endonucleases, vectors, ligation, propagation, selection
Type II restriction endos (Arber/phage/restriction/modification)
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Overview of DNA cloningEpisomal DNA (plasmid)
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EcoRV endonuclease: Mg++ dependent, blunt cutter, phosphodiesterase
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Prediction of site numberOrigin (200 bp, timing, plasmid specific)
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Plasmid cloning (libraries)
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Form of replica plating
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Replica plating
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Bacteriophage λ cloning vectors
48 Kb, ds DNA, linear
Remove genes not needed for phage replication
“Filler” genes
Capsids
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Bacterial artificial chromosomes (BACS) (John Carbon, UCSB, and others)
Cloning of 100-300Kb segments
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Yeast (eukaryotic) artificial chromosomes (YACS)
Yeast genome only 4X E.coli (14 x 106 bp)
Origin, CEN, telomeres, selectable markers
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Hybridization to identify clone/DNA segment
Selection vs screen
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Redundant probes (23 = 8)
Hybridization probe design
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Typical E.coli expression vector
Q: name two-three expression vectors sold by New England Biolabs, Invitrogen, Promega, Stratagene and their unique features
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Site directed mutagenesis (protein engineering, genetic engineering)…two approaches
Current method (Stratagene, Quickchange)
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9.2 From genes to genomes
•DNA libraries
•Contigs
•Sequence-tagged sites (STS)
•cDNAs/cDNA libraries
•Expression sequence tag (EST)
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Ordering of clones in DNA library
Markers
Contig: long contiguous segmentSTS: sequence-tagged site (markers)
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cDNA library from mRNAPoly T cellulose
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Reporter constructs, GFP, gene fusions, epitope tagsCellular, tissue, subcellular expression/localization
C.elegans, fusion protein expressed in specific neurons
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PCR, Mullis, Nobel prize, CEN article
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Cloning of amplified DNA
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RFLPs, SNPS
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The $1000 genomeGenomics, pharmacogenomics, toxicogenomicsPersonalized medicine, molecular diagnostics
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Viral DNASilenced by DNA methylation
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9.3 Genomes to proteomes
ProteomicsPhenotypicCellularMolecular
Comparative genomicsSynteny (conserved gene order)
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Cellular gene expressionGenetic networks 35,000 human genes1013 cellsHow controlled/regulated?
2D PAGE/MSGene chipsAffymetrixChip on Chip
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Genetic regulatory networks
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Recent method…Chip on Chip
Common strategies to “read the genome/proteome”
mRNA analysis
2D-PAGE and MS/MS
How to identify active regulatory parts of the genome which control transcription?
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Yeast two hybrid: identification of interacting proteins
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9.4 Genome alterations and biotech productsPlant bacterial parasite used for cloning (no plasmids)Agrobacterium (crown gall formation)
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Two plasmid approach to make recombinant plants
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Tobacco plant expressing firefly luciferase
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RoundupEngineered plants
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Retroviral vectors for mammalian cell cloning (gene therapy)Cell/tissue specificityEmasculated virusSilencing
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Spatial/temporal control over gene regulation: basic research/therapeutics of the future
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