DNABased Information Technologies

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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 24 ESB 1001330 PM (W.E. Palke
Memorial Lecture) Bruce Berne-  Columbia
University Hydrophobibity and Drying in Physical
and Biological Systems
BMSE SeminarFriday April 27, 2007300pmWebb
1100SILVIA CAVAGNEROUniversity of
Wisconsin-MadisonDepartment 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/modific
ation)
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Overview of DNA cloning Episomal DNA (plasmid)
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EcoRV endonuclease Mg dependent, blunt cutter,
phosphodiesterase
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Prediction of site number Origin (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 ? 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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Hybridization probe design
Redundant probes (23 8)
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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 segment STS
sequence-tagged site (markers)
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cDNA library from mRNA Poly T cellulose
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Reporter constructs, GFP, gene fusions, epitope
tags Cellular, 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 genome Genomics, pharmacogenomics,
toxicogenomics Personalized medicine, molecular
diagnostics
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9.3 Genomes to proteomes Proteomics Phenotypic Ce
llular Molecular
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Cellular gene expression Genetic networks 35,000
human genes 1013 cells How controlled/regulated?
2D PAGE/MS Gene chips Affymetrix Chip on Chip
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Genetic regulatory networks
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Common strategies to read the genome/proteome mR
NA 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 products Plant
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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Retroviral vectors for mammalian cell cloning
(gene therapy) Cell/tissue specificity Emasculated
virus Silencing
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Spatial/temporal control over gene regulation
basic research/therapeutics of the future
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