Ch 20 Biotechnology Overview The DNA Toolbox History

Ch. 20 Biotechnology

Overview: The DNA Toolbox History 1970’s Recombinant DNA 2001 Human Genome Project 10 years, $3 billion 2010 1200 Genomes Sequenced DNA microarray

20. 1 DNA Cloning Bacteria E. coli Plasmids Gene Cloning Pesticide/Herbicide Resistance Oil Spill Clean-up Dissolve Human Blood Clots Growth Hormone Plasmid

20. 1 DNA Cloning Restriction Enzymes Discovered in 1960’s Protect Bacteria from Viruses Very Specific-Restriction Site Restriction Fragments Sticky End-single sided DNA ligase-rejoins

20. 1 DNA Cloning Eukaryotic Genes Figure 20. 4 pg. 399 Cloning Vector-original plasmid Genetic Marker (ampicillin) Same restriction enzymes remove and insert gene of interest Plate to sort

20. 1 DNA Cloning DNA Libraries Shotgun approach Genomic Library Bacterial Artificial Chromosome (BAC) Larger Segments of DNA Complementary (c. DNA) library DNA

20. 1 DNA Cloning Screening Libraries Gene of Interest Nucleic Acid Hybridization Nucleic Acid Probe Expression Vector-inserts gene of interest into correct reading frame Electroporation-electrical pulse opens membrane

20. 1 DNA Cloning PCR Polymerase Chain Reactionthermocycler copies DNA fragments Use bacteria from hot springs Millions of copies in 24 hours

20. 2 Sequence, Expression & Function of Genes Gel Electrophoresis Southern Blotting

20. 2 Sequence, Expression & Function of Genes Dideoxy Chain Termination Northern Blotting Fig. 20. 12 pg. 408 4 th Generation 3 rd Generation 1980 Nobel Prize-Sanger “Sequencing by Synthesis” Used to study expression of single genes at a particular time in development.

20. 2 Sequence, Expression & Function of Genes 5 th Generation Reverse Transcriptasepolymerase reaction (RTPCR) c. DNA is used as a template in situ Hybridization Shows where in body genes are being expressed w/ fluorescent dyes

20. 2 Sequence, Expression & Function of Genes Interacting Genes DNA microarray assays (DNA chip) Shows how groups of genes are expressed as an organism develops in vitro mutagenesis Specific mutations introduced, used to produce organisms for scientific studies

20. 2 Sequence, Expression & Function of Genes RNA interference (RNAi) Blocks translation Turns specific genes off so that you determine its function. “knocking out” Prohibited in humans

20. 2 Sequence, Expression & Function of Genes Genome-wide Association Studies Large-scale analyses Used to study: heart disease, diabetes, autism, etc. Test for genetic markers Single nucleotide polymorphism (SNP)

20. 3 Stem Cell Research Plants Animals 1950’s Steward at Cornell University 1950’s Briggs & King w/ frogs Totipotent-cell that can differentiate into a specialized cell. 1970’s Gurdon-inversely related to age 1997 Scotland-Dolly the sheep 2007 Monkeys Many crop and decorative species: grapes, apples, orchids, etc.

20. 3 Stem Cell Research Problems w/ Animal Cloning Low success rate Defects Obesity, pneumonia & liver failure Caused by epigenetic changes in chromatin during development (methylation)

20. 3 Stem Cell Research Embryonic Stem Cells Blastula stage of development Reproduce indefinitely Pluripotent-can produce any type of cell Could be used to replace cell that don’t normally divide: nerve & heart Controversial-fetal tissue Adult Stem Cells Ex. Bone marrow Can only differentiate into limited cell types 2007 reprogramminginduced pluripotent stem cells (i. PS) Still in development, limited success

20. 4 Practical Applications Medical Identification of faulty genes Identification of pathogens “personalized medicine” Gene therapy-replacing defective genes, limited success. Ex. SCID bone marrow replacement Pharmaceuticals Cancer treatments Proteins HGH, Insulin, etc. Transgenic animals used to make medicines Forensic Evidence Genetic Profile, STRs

20. 4 Practical Applications Environmental Cleanup Ethics/Safety/Regulation Agriculture GMO debate Selective breeding Increased Disease Ti production resistance Wild varieties Allergies plasmid Environmental Insecticides/herbicide adaptation Personal information Designer babies