Biotechnology Chapter 20

1. BiotechnologyChapter 20

2. Gene technology

3. Biotechnology • Manipulation of organisms to make useful products

4. Genetic engineering • Manipulation of genes • Gene cloning: • Multiple copies of a single gene • Produce a specific product

5. Fig. 20-2 Cell containing geneof interest Bacterium 1 Gene inserted intoplasmid Bacterialchromosome Plasmid Gene ofinterest RecombinantDNA (plasmid) DNA of chromosome 2 Plasmid put intobacterial cell Recombinantbacterium 3 Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest Gene ofInterest Protein expressedby gene of interest Copies of gene Protein harvested Basic research andvarious applications 4 Basicresearchon protein Basicresearchon gene Gene used to alter bacteria for cleaning up toxic waste Gene for pest resistance inserted into plants Protein dissolvesblood clots in heartattack therapy Human growth hor-mone treats stuntedgrowth

6. Recombinant DNA • 1970’s • Combining genes from different sources • Even different species • Combined into single DNA • Example: Bacteria & mammal

7. Recombinant DNA • Genetically modified bacteria • Mass produce beneficial chemicals • Insulin • Growth hormone • Cancer drugs • Pesticides

8. Plasmid

9. Plasmid • Small separate circular DNA • Replicated same as main DNA • Foreign DNA added to plasmid • Replicated along with plasmid

10. Recombinant DNA • Nucleases: • Enzymes that degrade DNA • Restriction endonulceases: • Restriction enzymes • Cut DNA into fragments • Specific points

11. Recombinant DNA • Restriction sites: • Places where DNA is cut • Short DNA sequence

12. Recombinant DNA • Restriction enzyme • Recognizes short sequences in DNA • Cuts at these sequences • Staggered cut • Leaves single-stranded ends • Called “sticky ends”

13. Recombinant DNA D:\Chapter_20\A_PowerPoint_Lectures\20_Lecture_Presentation\2003RestrictionEnzymesA.html

17. Recombinant DNA • Sticky ends • Enables insertion of other DNA • DNA fragments from other sources • Match ends by base pairs (complementary sequences) • DNA ligase: • Enzyme combines ends • Forms a phosphodiester bond

20. Recombinant DNA (Process) • 1. Isolate gene of interest & bacterial plasmid • 2. Cut DNA & plasmid into fragments • 3. Mix DNA fragments with cut plasmid. • Fragment with gene of interest is inserted into the plasmid • 4. Recombinant plasmid is mixed with bacteria

21. Recombinant DNA (Process) • 5. Bacteria with recombinant DNA reproduce • 6. Isolate bacterial clones that contain gene of interest • Producing protein of interest • 7. Grow large quantities of bacteria that produce the protein

22. Recombinant DNA (Process) D:\Chapter_20\A_PowerPoint_Lectures\20_Lecture_Presentation\2004CloningAGeneA.html

23. Hummingbird cell TECHNIQUE Fig. 20-4-4 Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carryingplasmids RESULTS Colony carrying recombinant plasmid with disrupted lacZ gene Colony carrying non-recombinant plasmidwith intact lacZ gene One of manybacterial clones

24. Recombinant DNA • Vector: • DNA molecule that carry foreign DNA • Enters & replicates in the host • Plasmids & phages are common vectors • Phages are larger than plasmid • Can handle inserts up to 40 kilobases

25. PCR • Polymerase chain reaction • Amplify DNA • Makes large quantities of DNA • 1985

26. PCR • Heated • Denatured • DNA primer • Heat stable DNA polymerase • Makes DNA

27. Fig. 20-8 3 5 TECHNIQUE Targetsequence 3 5 Genomic DNA 1 5 3 Denaturation 5 3 2 Annealing Cycle 1yields 2 molecules Primers 3 Extension Newnucleo-tides Cycle 2yields 4 molecules Cycle 3yields 8 molecules;2 molecules(in whiteboxes)match targetsequence

28. Gel electrophoresis • Study DNA • Polymer (gel) • Restriction fragments • Separates DNA based on charge & size • Nucleic acids negative charge (Phosphates) • Migrate towards + end (red)

29. Fig. 20-9 TECHNIQUE Powersource Mixture ofDNA mol-ecules ofdifferentsizes – Cathode Anode + Gel 1 Powersource – + Longermolecules 2 Shortermolecules RESULTS

30. Fig. 20-10 Normal -globin allele Normalallele Sickle-cellallele 175 bp Large fragment 201 bp DdeI DdeI DdeI DdeI Largefragment Sickle-cell mutant -globin allele 376 bp 201 bp175 bp Large fragment 376 bp DdeI DdeI DdeI (a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles

31. Cloning • Multicellular organisms come from a single cell. • Offspring are identical

32. Cloning • 1950 • Carrots • Totipotent: • Mature cells that undifferentiated • Give rise to any type of cells • Common in plants

33. Cloning • Nuclear transplantation • Nucleus of an unfertilized/fertilized egg is removed • Replaced with nucleus of differentiated cell • Direct development of cell into tissues etc.

34. Cloning • Removed nuclei from an egg • Mammary cells • Fused with egg cells • Dolly, 1997, identical to mammary cell donor • Died prematurely age 6 • Arthritis & lung disease

35. TECHNIQUE Fig. 20-18 Mammarycell donor Egg celldonor 2 1 Egg cellfrom ovary Nucleusremoved Cells fused 3 Culturedmammary cells 3 Nucleus frommammary cell Grown inculture 4 Early embryo Implantedin uterusof a thirdsheep 5 Surrogatemother Embryonicdevelopment 6 Lamb (“Dolly”)genetically identical tomammary cell donor RESULTS

38. Fig. 20-19

39. Cloning • Few develop normally • Abnormalities • Epigenetic changes to the chromatin • More methylation of chromatin • Reprogram chromatin of differentiated cell

40. Stem cells • Started 1998 at UW • Early embryonic cells • Potential to become any type of cell • Master cell generates specialized cells • Such as muscle cells, bone cells, or blood cells

41. Stem cells • Embryos • Bone marrow • Umbilical cord blood • Blood stem cells • ?? Turn skin cells into embryonic stem cells • Therapeutic cloning

42. Embryonic stem cells Adult stem cells From bone marrowin this example Early human embryoat blastocyst stage(mammalian equiva-lent of blastula) Fig. 20-20 Cells generatingall embryoniccell types Cells generatingsome cell types Culturedstem cells Differentcultureconditions Differenttypes ofdifferentiatedcells Blood cells Nerve cells Liver cells

43. Medical applications • Genetic markers • Detect abnormal disease • SNP • Single nucleotide polymorphisms • Single base pair site where variation is found • RFLP • Restriction fragment length polymorphisms

44. Fig. 20-21 DNA T Normal allele SNP C Disease-causingallele

45. Medical applications • Gene therapy • Treat genetic defects • Alters person’s genes • 2 girls with rare blood disease • CF (vectors are viruses) • SCID (immune disorder) • Injected viral DNA with normal gene

46. Clonedgene Insert RNA version of normal alleleinto retrovirus. Fig. 20-22 1 Viral RNA Let retrovirus infect bone marrow cellsthat have been removed from thepatient and cultured. 2 Retroviruscapsid Viral DNA carrying the normalallele inserts into chromosome. 3 Bonemarrowcell frompatient Bonemarrow Inject engineeredcells into patient. 4

47. Medical applications • Transgenic animal • Gene from one animal is inserted into another • Goat milk protein anti-thrombin • Isolated from milk • “pharm” animals

48. Animals • Transgenic animals engineered for specific traits • Genetically create a racehorse • Not have to breed • Sheep with better wool??

50. Agricultural applications • Manipulate tomatoes • Do not ripen as fast • “Flavr-Savr” • Slows down ethylene production