2 OutlineRestriction EndonucleasesHost / Vector SystemsDNA LibrariesGenetic Engineering ExperimentWorking With Gene ClonesBiotechnologyMedical ApplicationsAgricultural ApplicationsRisk and Regulation
3 Restriction Endonucleases Restriction endonucleases recognize specific nucleotide sequences, and cleave DNA creating DNA fragments.Type I - simple cutsType II - dyad symmetryallows physical mappingallows recombinant molecules
4 Restriction Endonucleases Each restriction endonuclease has a specific recognition sequence and can cut DNA from any source into fragments.Because of complementarity, single-stranded ends can pair with each other.sticky endsfragments joined together with DNA ligase
7 Host / Vector SystemsDNA propagation in a host cell requires a vector that can enter the host and replicate.most flexible and common host is E. colitwo most commonly used vectors are plasmids and phagesviruses and artificial chromosomes also being probed for use
9 Using Vectors to Transfer Genes ChimerasOne of first recombinant genomes was a bacterial plasmid into which an amphibian ribosomal RNA gene was inserted.Viruses can also be used as vectors to insert foreign DNA into host cells.
12 DNA LibrariesA collection of DNA from a specific source in a form that can be propagated in a hostgenomic library - representation of the entire genome in a vectorcDNA library is limited to expressed genes isolated by reverse transcriptaseisolated from retroviruses
15 Genetic Engineering Experiment Four stagesDNA cleavagerestriction endonuclease cleaves source DNA into fragmentsproduction of recombinant DNADNA fragments inserted into plasmids or viral vectorscloning
16 Genetic Engineering Experiment Screeningclones with DNA fragment of interest identified from clone librarypreliminary screening - eliminate any clones without a vector and clones with vectors that do not contain DNAemploy vector with gene for antibiotic resistance and lac Z’ geneexpose to growth medium
17 Genetic Engineering Experiment Secondary screening (gene of interest)hybridization - cloned genes form base pairs with complementary sequences on another nucleic acid (probe)grow on agar then transfer to filter pressed on coloniestreat filter with radioactive probe, and perform autoradiography
22 Working With Gene Clones Identifying DNA: Southern blottingsample DNA cleaved into restriction fragments, and spread apart by gel electrophoresisgel blotted with sheet of nitrocelluloseprobe of purified, single-stranded DNA poured over sheetif radioactive probe used, band of radioactivity appears where probe hybridized with complementary fragment
27 BiotechnologyMedical applicationspharmaceuticalsintroduction of protein-encoding genesatrial peptides - high blood pressure and kidney failuretissue plasminogen activator - dissolving blood clotsgene therapyadd working copies of single defective gene
28 Medical ApplicationsPiggyback vaccinesproduce subunit vaccines against virusesherpeshepatitisDNA vaccinecellular immune response
30 Agricultural Applications Ti plasmid has been early successful vector.nitrogen fixationintroduce genes that allow crops to fix nitrogenreduce need for fertilizerherbicide resistanceinsert genes encoding for proteins making crops resistant to herbicidewidespread herbicide use possible
36 Risk and RegulationQuestionsHow do we measure the potential risks of genetically modified crops ?Is eating genetically modified food dangerous ?Are genetically modified crops harmful to the environment ?Should we label genetically modified foods ?
37 SummaryRestriction EndonucleasesHost / Vector SystemsDNA LibrariesGenetic Engineering ExperimentWorking With Gene ClonesBiotechnologyMedical ApplicationsAgricultural ApplicationsRisk and Regulation