Chapter 9:DNA-Based Information Technologies

DNA cloning: the basics

Genes to genomes

Genomes to proteomes

Genome alterations/new biotechnology products

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

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)

Overview of DNA cloningEpisomal DNA (plasmid)

EcoRV endonuclease: Mg++ dependent, blunt cutter, phosphodiesterase

Prediction of site numberOrigin (200 bp, timing, plasmid specific)

Plasmid cloning (libraries)

Form of replica plating

Replica plating

Bacteriophage λ cloning vectors

48 Kb, ds DNA, linear

Remove genes not needed for phage replication

“Filler” genes

Capsids

Bacterial artificial chromosomes (BACS) (John Carbon, UCSB, and others)

Cloning of 100-300Kb segments

Yeast (eukaryotic) artificial chromosomes (YACS)

Yeast genome only 4X E.coli (14 x 106 bp)

Origin, CEN, telomeres, selectable markers

Hybridization to identify clone/DNA segment

Selection vs screen

Redundant probes (23 = 8)

Hybridization probe design

Typical E.coli expression vector

Q: name two-three expression vectors sold by New England Biolabs, Invitrogen, Promega, Stratagene and their unique features

Site directed mutagenesis (protein engineering, genetic engineering)…two approaches

Current method (Stratagene, Quickchange)

9.2 From genes to genomes

•DNA libraries

•Contigs

•Sequence-tagged sites (STS)

•cDNAs/cDNA libraries

•Expression sequence tag (EST)

Ordering of clones in DNA library

Markers

Contig: long contiguous segmentSTS: sequence-tagged site (markers)

cDNA library from mRNAPoly T cellulose

Reporter constructs, GFP, gene fusions, epitope tagsCellular, tissue, subcellular expression/localization

C.elegans, fusion protein expressed in specific neurons

PCR, Mullis, Nobel prize, CEN article

Cloning of amplified DNA

RFLPs, SNPS

The $1000 genomeGenomics, pharmacogenomics, toxicogenomicsPersonalized medicine, molecular diagnostics

Viral DNASilenced by DNA methylation

9.3 Genomes to proteomes

ProteomicsPhenotypicCellularMolecular

Comparative genomicsSynteny (conserved gene order)

Cellular gene expressionGenetic networks 35,000 human genes1013 cellsHow controlled/regulated?

2D PAGE/MSGene chipsAffymetrixChip on Chip

Genetic regulatory networks

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?

Yeast two hybrid: identification of interacting proteins

9.4 Genome alterations and biotech productsPlant bacterial parasite used for cloning (no plasmids)Agrobacterium (crown gall formation)

Two plasmid approach to make recombinant plants

Tobacco plant expressing firefly luciferase

RoundupEngineered plants

Retroviral vectors for mammalian cell cloning (gene therapy)Cell/tissue specificityEmasculated virusSilencing

Spatial/temporal control over gene regulation: basic research/therapeutics of the future