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

2. Link to Cohen Boyer Early Genetic Expt

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

4. 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)

5. 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)

6. 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

7. Figure 18.x7 E. coli

8. Figure 18.x10 Plasmids

9. Link to Restriction Enzymes

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

11. Link to Gene cloning animation

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

13. 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

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

15. 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

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

17. 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

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

19. 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