After studying chapter 23 you will be able to: Know the general properties of a plasmid, know the general properties of restriction endonucleases, know the general properties of restriction endonucleases, know why we transform plasmids into bacteria, know how a cDNA Library is constructed, know how to make cDNA, know why we need to make cDNA, | Chapter 23 (Part 1) Recombinant DNA Technology Cloning Vector Required features Origin of replication Selectable marker Screenable marker for recombinant molecules Cloning sites Restriction Modification System AAGATGCGAATTCGTACA AAGATGCGAATTCGTACA * * AAGATGCGAATTCGTACA * * DNA methylase Restriction endonuclease AAGATGCGAATTCGTACA AAGATGCGAATTCGTACA AAGATGCG AATTCGTACA DNA methylase Restriction endonuclease 5’ ATGCGAATTCCGGTT 3’ 3’ TACGCTTAAGGCCTT 5’ 5’-ATGCG-3’ 5’-AATTCCGGTT-3’ 3’-TACGCTTAA-5’ 3’-GGCCTT-5’ EcoR1 5’ ATGCGATATCCGGTT 3’ 3’ TACGCTATAGGCCTT 5’ 5’-ATGCGAT-3’ 5’-ATCCGGTT-3’ 3’-TACGCTA-5’ 3’-TAGGCCTT-5’ EcoRV Sticky-end cutter Blunt-end cutter T4 DNA Ligase Transformation All of the previous steps were performed in vitro. We have generated a very small amount of a recombinant plasmid Need to amplify in bacteria to get enough to work with. Transformation – process to mobilize DNA into bacterial host Select for transformed bacteria on specific antibiotic that corresponds to the antibiotic resistance gene present on the plasmid How to produce a recombinant protein 10 to 70% of cellular protein to 1% of cellular protein cDNA cDNA Library cDNA DNA hydridization screening for specific gene Requires that you know something about the gene sequence Can get sequence information form purified protein Product name Protein type Application Company Adagen (Adenosine deaminase ) An enzyme Severe combined immunodeficiency disease (SCID) Enzon Genotropin (Recombinant growth hormone) A hormone Growth hormone deficiency (GHD) in children Pharmacia & Upjohn Humalog (Recombinant human insulin) A hormone Diabetes Eli Lilly Nabi-HB (Anti-Hepatitis B) An antibody Hepatitis-B Nabi Novo Seven (Recombinant coagulation factor VIIa) A modified factor Hemophillia patients with inhibitors Novo Nordisk Ontak (Diphtheria toxin-interleukin-2) A fusion protein Cutaneous T-cell lymphoma (CTCL) Ligand Pharmaceuticals Roferon-A (Recombinant interferon alfa-2a) A modifier Hairy cell leukemia or AIDS-related Kaposis sarcoma Hoffmann-La Roche Genetic Modification of Higher Organisms Can introduce gene into animals and plants These modified organism are powerful research tools to study the effect of a specific gene product on metabolism, development etc . Has also been used to develop improved agricultural products Genetically Engineered Salmon Is Bigger Better? | Chapter 23 (Part 1) Recombinant DNA Technology Cloning Vector Required features Origin of replication Selectable marker Screenable marker for recombinant molecules Cloning sites Restriction Modification System AAGATGCGAATTCGTACA AAGATGCGAATTCGTACA * * AAGATGCGAATTCGTACA * * DNA methylase Restriction endonuclease AAGATGCGAATTCGTACA AAGATGCGAATTCGTACA AAGATGCG AATTCGTACA DNA methylase Restriction endonuclease 5’ ATGCGAATTCCGGTT 3’ 3’ TACGCTTAAGGCCTT 5’ 5’-ATGCG-3’ 5’-AATTCCGGTT-3’ 3’-TACGCTTAA-5’ 3’-GGCCTT-5’ EcoR1 5’ ATGCGATATCCGGTT 3’ 3’ TACGCTATAGGCCTT 5’ 5’-ATGCGAT-3’ 5’-ATCCGGTT-3’ 3’-TACGCTA-5’ 3’-TAGGCCTT-5’ EcoRV Sticky-end cutter Blunt-end cutter T4 DNA Ligase Transformation All of the previous steps were performed in vitro. We have generated a very small amount of a recombinant plasmid Need to amplify in bacteria to get enough to work with. Transformation – process to mobilize DNA into bacterial host Select for transformed bacteria on specific antibiotic that corresponds to .
