Recombinant DNA Recombinant DNA – DNA that contains a gene inserted from a different organism (or a modified gene from the host)

Link to Cohen Boyer Early Genetic Expt

DNA Interactive Genetics to Genomics- Advances and Applications Birth of Genetic Engineering Impact of Cloning Insulin Production Watson – Cotton Plants

Benefits and Risks of Recombinant DNA Large scale production of desired proteins (examples, Insulin production, TPA) Provide organism with new desirable traits (Examples, plants resistant to frost or insects) Cure genetic diseases by insertion of corrected copy of gene RISKS Modified organisms may have survival advantages to outcompete native organisms thereby changing ecological balances Production of bacteria/viruses with dangerous properties Moral/ethical/religious (Humans should not play God)

Cloning Clone – identical copy Gene Cloning – make many copies of a gene Cell Cloning – grow identical copies of a cell Organism Cloning – create identical copies of an organism; (all cloned organism contain identical DNA sequences)

Figure 20.1 An overview of how bacterial plasmids are used to clone genes Gene Cloning – obtaining many copies of a gene by inserting the gene into a bacterial host and allowing cells to multiply

Figure 18.x7 E. coli

Figure 18.x10 Plasmids

Link to Restriction Enzymes

Figure 20.2 Using a restriction enzyme and DNA ligase to make recombinant DNA

Link to Gene cloning animation

Figure 20.3 Cloning a human gene in a bacterial plasmid: a closer look (Layer 1) Use cDNA

Figure 20.5 Making complementary DNA (cDNA) for a eukaryotic gene cDNA = gene from eukaryote with introns removed Reverse transcriptase= enzyme isolated from retrovirus that transcribes DNA from RNA template

Figure 20.3 Cloning a human gene in a bacterial plasmid: a closer look (Layer 2)

Bacterial Transformation Bacteria takes in foreign DNA from outside cell Advantage to organism: may obtain useful DNA such as antibiotic resistance Can be induced by electric shock to membrane or by using hot and cold shock in a CaCl2 bath. Link to transformation animation

Figure 20.3 Cloning a human gene in a bacterial plasmid: a closer look (Layer 3)

Methods of Identifying Cell Colonies with Gene of Interest Antibiotic resistance – recombinant insert contains gene for resistance antibiotics such as ampicillin Recombinant insert breaks apart β-galactosidase gene; Add X-Galactose → produces blue product when hydrolyzed; cells WITH DNA of interest remain WHITE, cells without desired DNA insert will be blue. Identify gene product – examples :Green fluorescent protein or luciferase; cells of interest will glow Use radioactive complementary DNA probes to search for DNA sequence

Figure 20.4 Using a nucleic acid probe to identify a cloned gene

Fig. 20-5 Examples of Gene Libraries – collections of cells containing inserted genes Foreign genome cut up with restriction enzyme Large insert with many genes Large plasmid or BAC clone Recombinant phage DNA Bacterial clones Recombinant plasmids Phage clones (a) Plasmid library (b) Phage library (c) A library of bacterial artificial chromosome (BAC) clones