Biotechnology

1. Biotechnology Dolly repairstemcell.files.wordpress.com

2. Potential Uses for Cloned Genes • to produce a protein product • plasminogen activator • to endow an organism with a metabolic capability • engineered bacteria that degrade oil spills • create more copies of the gene for further study

3. Genetic Engineering Stages • 1. Isolate gene • cleavage of plasmid • 2. Recombinant-DNA • 3. Clone • * Screen for quality • 4. Harvest • Gene • Protein

4. 1. Isolate Gene • Isolate the desired gene from the genome • the desired gene will be cut with restriction endonucleases (enzymes) desired gene to be isolated

5. Cleavage • cut DNA of host with restriction endonucleases (enzymes) • several hundred RE’s exist discovered 1960s • produce sticky ends or blunt ends

6. Sticky Ends Escherichia coli Eco RI sticky end

7. Blunt ends Haemophilus aegyptius Hae III G G C C C C G G G G C C C C G G

8. 2. Recombinant DNA • splice desired gene into host DNA, DNA ligase seals the strands • Vector, generally • plasmid of bacteria (prok) or yeast (euk) • viral DNA (not for harvesting protein) plasmid with recombinant DNA

9. 3. Clone produce a cell line in which all members have identical copies of a particular gene Screen Choose cells that carry desired gene & eliminate those cells that do not carry desired gene

10. 4. Harvest (or Isolate) • harvest protein • harvest gene genetic harvesting protein harvesting protein molecules copies of the gene pest resistant gene oil eating bacteria dissolving clot protein human growth hormone

11. Cloning reverse transcriptase plus mRNAs mRNAs • Directly from an organism • complementary DNA • made from mRNA template through reverse transcription (cDNA) • Reverse transcriptase can be used to make smaller cDNA libraries • These contain only the genes that are transcribed by a particular type of cell • recognized by the addition of a RE recognition sequence to it mRNA is degraded by an enzyme DNA polymerase synthesizes the 2nd strand cDNA

12. Genomic Libraries • “Book,” a clone containing a foreign DNA • Plasmid library (bacterial, yeast) • Phage library (virus) • Bacterial Artificial Chromosome library

13. Plasmid Library • Copies of DNA fragments can be stored in a cloned bacterial plasmid foreign genome Each one of these is considered a "book" recombinant plasmid bacterial clones

14. Phage Library • DNA fragments can be stored in a cloned phage • each phage type is considered a "book" foreign genome recombinant phage "book" phage clones

15. Bacterial Artificial Chromosome (BAC) Library Copies of multiple DNA fragments can be stored in a bacterial artificial chromosome BAC clone plasmid with many genes each clone occupies one well

16. Gene products Product Made in Use human insulin E. coli diabetes human growth hormone E. coli growth defects epidermal growth factor E. coli burns, ulcers interleukin-2 E. coli possibly cancer bovine growth hormone E. coli improving weight gain cellulase E. coli breakdown of cellulose Taxol E. coli ovarian cancer hepatitis B vaccine S. cerevisiae prevents hepatitis erythropoietin mammalian cells anemia factor VIII mammalian cells hemophilia tissue plasminogen activator mammalian cells heart attacks

17. Other Examples • "golden rice" • genetically modified • rich in beta-carotene • prevents blindness news.bbc.co.uk

18. papaya's ring spot disease • gene was introduced to control the plague http://www2.dpi.qld.gov.au

19. Human Genome Project • Collaborative effort to map and sequence entire human genome • Began 1990 • 4 goals • genetic (linkage) mapping • physical mapping • sequencing • analyzing the genomes of other species

20. Genetic Mapping of the Human Genome • to locate genetic markers spaced evenly throughout the chromosomes • to make it easier to find other loci

21. Physical Mapping of the Human Genome • cutting chromosomes into identifiable fragments then determining their order on the chromosome

22. Sequencing the Human Genome • determining the exact nucleotide pairs • haploid set of human chromosomes contains approximately 3 billion nucleotide pairs • Genbank • Database where DNA sequences have been deposited • publicly available via the Internet • final draft, 2004 (over 99% of genome was determined) • remain a few 100 gaps of unknown sequences that require special methods to figure out

23. Analyzing Gene Expression • Analyze genomes of other important species for genetic engineering

24. Stem Cells • unspecialized • blastula cells • pluripotent • adult stem cells • gives rise to specific types of cells • bone marrow blood cells

25. Applications • Medical • Diagnosis • Human Gene therapy • Pharmaceutical products • animal and plant application • gold rice • salinity resistant gene • Environmental • biofuel • oil cleaning bacteria • Forensic evidence • The Innocence Project • conviction of guilty

26. Genomes of other species and H. sapiens • Bacteria • H. influenzae 1,700 1995 • E. coli 4,400 1997 • Fungi • S. cerevisiae 6,200 1996 • Plants • Oryza sativa (rice) 60,000 2002 • Animals • D. melanogaster 13,700 2000 • Mus musculus 22,000 2001 • Rattus norvegius 25,000 2004 • H. sapiens 21,000 2003

27. Ethical Issues • Should we engineer new genotypes for individuals with anomalies? • diabetes, CF, immune deficiencies, MD, stunted growth, sickle-cell disease • myopia, altering personalities, increase length of life

28. Should we engineer human germ cells? • If they are carrying abnormal genes • eugenics - deliberate effort to control the genetic makeup of human populations • color of eyes • color of skin • color of hair

29. We have technology to test for diseases for which there is no cure and sometimes no treatment. (Ex. Huntington’s disease, breast cancer) • Would you want to be tested?

30. Who should have right to examine someone’s genetic info? • How should that info be used? • Should a person’s genome be a factor in determining eligibility for a job or insurance The End