Ch.20 Biotechnology

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

20.1 DNA Cloning Bacteria E. coli  Plasmids  Gene Cloning  Pesticide/Herbicide Resistance  Oil Spill Clean-up  Dissolve Blood Clots  Human 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 DNA (cDNA) library

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 Reaction-thermocycler copies DNA fragments  Use bacteria from hot springs  Millions of copies in 24 hours

20.2 Sequence, Expression & Function of Genes Gel ElectrophoresisSouthern Blotting

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

20.2 Sequence, Expression & Function of Genes 5 th Generation  Reverse Transcriptase- polymerase reaction (RT- PCR)  cDNA 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  1950’s Steward at Cornell University  Totipotent-cell that can differentiate into a specialized cell.  Many crop and decorative species: grapes, apples, orchids, etc. Animals  1950’s Briggs & King w/ frogs  1970’s Gurdon-inversely related to age  1997 Scotland-Dolly the sheep  2007 Monkeys

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 reprogramming- induced pluripotent stem cells (iPS)  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  Agriculture  Selective breeding  Increased production  Disease resistance  Ti plasmid  Environmental adaptation  Ethics/Safety/Regulation  GMO debate  Insecticides/herbicide resistance  Wild varieties  Allergies  Personal information  Designer babies