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Understanding Recombinant DNA Technology: Cloning Vectors and Applications

This chapter explores the essential features of cloning vectors used in recombinant DNA technology. Key components include the origin of replication, selectable and screenable markers, and various cloning sites. It details the roles of restriction endonucleases and DNA ligase in constructing recombinant plasmids, as well as the transformation process to amplify the plasmid in bacterial hosts. The chapter also highlights applications of recombinant proteins, including treatments for severe combined immunodeficiency (SCID), growth hormone deficiency, diabetes, and genetic modification in agriculture.

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Understanding Recombinant DNA Technology: Cloning Vectors and Applications

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  1. Chapter 23 (Part 1) Recombinant DNA Technology

  2. Cloning Vector Required features • Origin of replication • Selectable marker • Screenable marker for recombinant molecules • Cloning sites

  3. AAGATGCGAATTCGTACA AAGATGCGAATTCGTACA DNA methylase DNA methylase * * AAGATGCGAATTCGTACA AAGATGCGAATTCGTACA Restriction endonuclease Restriction endonuclease * * AAGATGCG AATTCGTACA AAGATGCGAATTCGTACA Restriction Modification System

  4. 5’ ATGCGAATTCCGGTT 3’ 3’ TACGCTTAAGGCCTT 5’ EcoR1 5’-ATGCG-3’ 5’-AATTCCGGTT-3’ 3’-TACGCTTAA-5’ 3’-GGCCTT-5’ 5’ ATGCGATATCCGGTT 3’ 3’ TACGCTATAGGCCTT 5’ EcoRV 5’-ATGCGAT-3’ 5’-ATCCGGTT-3’ 3’-TACGCTA-5’ 3’-TAGGCCTT-5’ Sticky-end cutter Blunt-end cutter

  5. T4 DNA Ligase

  6. 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

  7. How to produce a recombinant protein 0.1 to 1% of cellular protein 10 to 70% of cellular protein

  8. cDNA

  9. cDNA Library cDNA

  10. DNA hydridization screening for specific gene • Requires that you know something about the gene sequence • Can get sequence information form purified protein

  11. 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 Kaposi's sarcoma Hoffmann-La Roche

  12. 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

  13. Genetically Engineered Salmon Is Bigger Better?

  14. http://www.agwest.sk.ca/sabic_index_tp.shtml

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