2. Chapter 17 – 18 Biotechnology: Genomics & DNA Technology

3.  Sanger method  determine the base sequence of DNA  dideoxynucleotides  ddATP, ddGTP, ddTTP, ddCTP  missing O for bonding of next nucleotide  terminates chain DNA Sequencing

4.  Sanger method  synthesize complementary DNA strand in vitro  in each tube:  “normal” N-bases  dideoxy N-bases  ddA, ddC, ddG, ddT  DNA polymerase  primer  buffers & salt 2 1 3 4 2

5. Fred Sanger 1978 | 1980 This was his 2nd Nobel Prize!!  1st was in 1958 for the structure of insulin

6. Advancements to Sequencing  Fluorescent tagging  no more radioactivity  all 4 bases in 1 lane  each base a different color  Automated reading

7. Advancements to Sequencing  Fluorescent tagging sequence data  Computer read & analyzed

8. Raw Genome Data

9. Automated Sequencing Machines  Really BIG labs!

10. Human Genome Project  U.S government project  begun in 1990  estimated to be a 15 year project  DOE & NIH  initiated by Jim Watson  led by Francis Collins  goal was to sequence entire human genome  3 billion base pairs  Celera Genomics  Craig Venter challenged gov’t  would do it faster, cheaper  private company

11. Different Approaches 3. Assemble DNA sequence using overlapping sequences. “map-based method” gov’t method “shotgun method” Craig Venter’s method 1. Cut DNA from entire chromosome into small fragments and clone. 2. Sequence each segment & arrange based on overlapping nucleotide sequences. 1. Cut DNA from entire chromosome into small fragments and clone. 2. Sequence each segment & arrange based on overlapping nucleotide sequences. 1.Cut chromosomal DNA segment into fragments, arrange based on overlapping nucleotide sequences, and clone fragments. 2. Cut and clone into smaller fragments. 1.Cut chromosomal DNA segment into fragments, arrange based on overlapping nucleotide sequences, and clone fragments. 2. Cut and clone into smaller fragments.

12. Human Genome Project On June 26, 2001, HGP published the “working draft” of the DNA sequence of the human genome. Historic Event!  blueprint of a human  the potential to change science & medicine

13. How does our genome stack up? Organism Genome Size (bases) Estimated Genes Human (Homo sapiens) 3 billion30,000 Laboratory mouse (M. musculus) 2.6 billion30,000 Mustard weed (A. thaliana) 100 million25,000 Roundworm (C. elegans) 97 million19,000 Fruit fly (D. melanogaster) 137 million13,000 Yeast (S. cerevisiae) 12.1 million6,000 Bacterium (E. coli) 4.6 million3,200 Human Immunodeficiency Virus (HIV) 97009

14. GenBank Database of genetic sequences gathered from research Publicly available!

15. Organizing the Data

16. And we didn’t stop there…

17. Interspersed Repetitive DNA  Repetitive DNA is spread throughout genome  interspersed repetitive DNA (SINEs Short INterspersed Elements) make up 25-40% of mammalian genome  in humans, at least 5% of genome is made of a family of similar sequences called, Alu elements (PV92 anyone?!)  300 bases long  Alu is an example of a "jumping gene" called a transposon; a DNA sequence that "reproduces" by copying itself & inserting into new chromosome locations

18. Rearrangements in the Genome  Transposons  transposable genetic element  piece of DNA that can move from one location to another in cell’s genome One gene of an insertion sequence codes for transposase, which catalyzes the transposon’s movement. The inverted repeats, about 20 to 40 nucleotide pairs long, are backward, upside-down versions of each other. In transposition, transposase molecules bind to the inverted repeats & catalyze the cutting & resealing of DNA required for insertion of the transposon at a target site.

19. Transposons Insertion of transposon sequence in new position in genome Insertion sequences cause mutations when they happen to land within the coding sequence of a gene or within a DNA region that regulates gene expression.

20. Transposons  Barbara McClintock  discovered 1st transposons in Zea mays (corn) in 1947 1947 | 1983

22. Retrotransposons  Transposons actually make up over 50% of the corn (maize) genome & 10% of the human genome. Most of these transposons are retrotransposons, transposable elements that move within a genome by means of RNA intermediate, transcript of the retrotransposon DNA.

23. Families of Genes  Human globin gene family  evolved from duplication of common ancestral globin gene Different versions are expressed at different times in development allowing hemoglobin to function throughout life of developing animal

24. Hemoglobin Differential expression of different beta globin genes ensures important physiological changes during human development.

25. The BIG Questions…  How can we use our knowledge of DNA to:  diagnose disease or genetic defect?  cure disease or genetic defect?  change/improve organisms?  What are the techniques & applications of biotechnology?  direct manipulation of genes for practical purposes

26. Biotechnology  Genetic manipulation of organisms is not new  humans have been doing this for thousands of years  plant & animal breeding

27. Evolution & Breeding of Food Plants Evolution of Zea mays from ancestral teosinte (left) to modern corn (right). The middle figure shows possible hybrids of teosinte & early corn varieties artificial selection!

28. Evolution & Breeding of Food Plants  “Descendants” of the wild mustard  Brassica genus artificial selection!

29. Animal Husbandry / Breeding artificial selection!

30. Biotechnology Today  Genetic Engineering  direct manipulation of DNA  if you are going to engineer DNA & genes & organisms, then you need a set of tools to work with  this unit is a survey of those tools… Our tool kit…

31. Bioengineering Tool Kit  Basic Tools  restriction enzymes  ligase  plasmids for gene cloning  gel electrophoresis  Advanced Tools  PCR  DNA sequencing  Southern blotting  DNA libraries / probes  microarrays

32. Cut, Paste, Copy, Find…  Word processing metaphor…  cut (Ctrl + X)  restriction enzymes  paste (Ctrl + V)  ligase  copy (Ctrl + C)  via plasmids  bacteria  transformation  via PCR  find (Ctrl + F)  Southern blotting  probes

33. Cutting DNA  Restriction enzymes  restriction endonucleases  discovered in 1960s  evolved in bacteria to cut up foreign DNA (“action restricted to foreign DNA”)  protection against viruses & other bacteria  bacteria protect their own DNA by methylation & by not using the base sequences recognized by the enzymes in their own DNA

34. Paste DNA  Sticky ends allow:  H bonds between complementary bases to anneal  Ligase  enzyme “seals” strands  bonds sugar- phosphate bonds  covalent bond of DNA backbone

35. AATTC GAATTC G G G G G CTTAAG GAATTC CTTAAG CTTAA CTTAAG DNA ligase joins the strands. DNA ligase joins the strands. DNA Sticky ends (complementary single-stranded DNA tails) Sticky ends (complementary single-stranded DNA tails) Recombinant DNA molecule AATTC G G CTTAA Biotech Use of Restriction Enzymes Restriction enzyme cuts the DNA Restriction enzyme cuts the DNA Add DNA from another source cut with same restriction enzyme

36. Application of Recombinant DNA  Combining sequences of DNA from 2 different sources into 1 DNA molecule  often from different species  human insulin gene in E. coli (humulin)  frost resistant gene from Arctic fish in strawberries  “Roundup-ready” bacterial gene in soybeans  BT bacterial gene in corn GFP  jellyfish glow gene in Zebra “Glofish” – GFP!

37. GFP Development of GFP  Shimomura, Chalfie, Tsien  discovery, isolation, and purification of GFP and many fluorescent analogs 1961, 1994 | 2008 Osamu Shimomura Martin Chalfie Roger Tsien

38. Cut, Paste, Copy, Find…  Word processing metaphor…  cut  restriction enzymes  paste  ligase  copy  plasmids  bacteria  transformation  PCR (chapter 11)  find  Southern blotting  probes

39. Plasmids Trust me… this will be important!

40. Plasmids  Plasmids  small supplemental circles of DNA  5000 - 20,000 base pairs  self-replicating  carry extra genes  2-30 genes  can be exchanged between bacteria  bacterial ‘sex’!!  rapid evolution  antibiotic resistance  can be imported from environment

41. Transformation  Bacteria are opportunists  pick up naked foreign DNA wherever it may be hanging out  have surface transport proteins that are specialized for the uptake of naked DNA  import bits of chromosomes from other bacteria  incorporate the DNA bits into their own chromosome  express new gene  form of recombination Who’s experiment documented this?

42. Swapping DNA  Genetic recombination by trading DNA 132 arg+ trp- arg- trp+ minimal media

43. Plasmids & Antibiotic Resistance  Resistance is futile?  1 st recognized in 1950s in Japan  bacterial dysentery not responding to antibiotics  worldwide problem now  resistant genes are on plasmids that are swapped between bacteria

44. Copy DNA  Plasmids  small, self-replicating circular DNA molecules  insert DNA sequence into plasmid  vector = “vehicle” into organism  transformation  insert recombinant plasmid into bacteria  bacteria make lots of copies of plasmid  grow recombinant bacteria on agar plate  clone of cells = lots of bacteria  production of many copies of inserted gene DNA  RNA  protein  trait

45. Biotechnology  Used to insert new genes into bacteria  example: pUC18  engineered plasmid used in biotech antibiotic resistance gene on plasmid is used as a selective agent

46. Biotechnology  Used to insert new genes into bacteria  example: pUC18  engineered plasmid used in biotech antibiotic resistance gene on plasmid is used as a selective agent

47. Selection for Plasmid Uptake  Ampicillin becomes a selecting agent  only bacteria with the plasmid will grow on amp plate LB/amp plateLB plate all bacteria grow only transformed bacteria grow

48. Recombinant Plasmid  Antibiotic resistance genes as a selectable marker  Restriction sites for splicing in gene of interest Selectable marker  Plasmid has both “added” gene & antibiotic resistance gene  If bacteria don’t pick up plasmid then “die” on antibiotic plates  If bacteria pick up plasmid then survive on antibiotic plates  selecting for successful transformation selection GFP

49. Gene Cloning

50. Cut, Paste, Copy, Find…  Word processing metaphor…  cut  restriction enzymes  paste  ligase  copy  plasmids  bacteria  transformation  PCR (chapter 11)  find  Southern blotting  probes

51. Any Questions?