1. PowerLecture: Chapter 16
Studying and Manipulating Genomes

2. Golden Rice, or Frankenfood?
Ordinary daffodil
Fig. 16-1a, p.242

3. Impacts, Issues: Golden Rice, or Frankenfood?
124 million children around the world have vitamin A deficiencies
Golden rice
–Rice plants engineered with genes from daffodils allowing it to produce beta-carotine in its seeds (rice)
–Beta carotine is the precursor to Vitamin A
Rice is the main food for 3 billion people

4. Golden Rice, or Frankenfood?
2nd generation
1st generation
Regular rice
Fig. 16-1b, p.242

5. Impacts, Issues: Golden Rice, or Frankenfood?
Many crops plants have been modified, including corn, beets, potatoes, and cotton
Potentially less harmful to the environment than current agricultural practices

6. Golden Rice, or Frankenfood?
Today's corn
Ancestral corn
p.243

7. Discovery of Restriction Enzymes
Hamilton Smith was studying how Haemophilus influenzae defend themselves from bacteriophage attack
Discovered bacteria have an enzyme that chops up viral DNA

8. Specificity of Cuts Restriction enzymes cut DNA at a specific sequence
Number of cuts made in DNA will depend on number of times the “target” sequence occurs

9. G another DNA fragment 5’ 3’ one DNA fragment C T A 3’ 5’ G C T A nick
DNA ligase action G C T A 3’ 5’
Fig. 16-2, p.244

10. Terms DNA ligase – seals cuts in DNA
Recombinant DNA – any molecule consisting of base sequences from 2 or more organisms of the same or different species
Cloning vector – a plasmid that has accepted foreign DNA and can be slipped into host

11. Using Plasmids Plasmid is small circle of bacterial DNA
Foreign DNA can be inserted into plasmid
Forms recombinant plasmids
Plasmid is a cloning vector
Can deliver DNA into another cell

12. Plasmids
Fig. 16-3a, p.244

13. Using Plasmids Fig. 16-4, p.245 e The DNA fragments and the plasmid
DNA are mixed with DNA ligase.
a A restriction enzyme
cuts a specific base
sequence everywhere it occurs in DNA.
b The DNA fragments
have sticky ends.
f The result? A collection of recombinant plasmids
that incorporate foreign DNA fragments.
c The same enzyme cuts the same sequnece in plasmid DNA.
d The plasmid DNA also has sticky ends
g Host cells that can divide rapidly take up the recombinant plasmids.
Fig. 16-4, p.245

14. Making cDNA
Use reverse transcriptase onto one strand of complementary DNA (cDNA)‏
DNA polymerase strips RNA bases
Copies cDNA into a second strand
Fig. 16-5, p.245

15. Gene Libraries Bacteria that contain different cloned DNA fragments
Genomic library
cDNA library

16. Use of a Probe
Colonies on plate
You want to find which bacteria in a library contain a specific gene
Need a probe for that gene
A radioisotope- labeled piece of DNA
Will base-pair with gene of interest
Cells adhere
to filter
Cells are lysed;
DNA sticks
to filter
Probe is
added
Location where probe binds forms
dark spot on film, indicates colony with gene

17. Making a Probe Make a primer if the sequence is known.
If not usually DNA from closely related species

18. Amplifying DNA
Fragments can be inserted into fast-growing microorganisms
Polymerase chain reaction (PCR)

19. Polymerase Chain Reaction
Sequence to be copied is heated
Primers are added and bind to ends of single strands
DNA polymerase uses free nucleotides to create complementary strands
Doubles number of copies of DNA
Animation

20. Polymerase Chain Reaction
Double-stranded
DNA to copy
DNA heated to
90°– 94°C
Primers added to
base-pair with
ends
Mixture cooled;
base-pairing of
primers and ends
of DNA strands
DNA polymerases
assemble new
DNA strands
Stepped Art
Fig. 16-6, p. 256

21. Polymerase Chain Reaction
Mixture heated again; makes all DNA fragments unwind
Mixture cooled; base-pairing between primers and ends of single DNA strands
DNA polymerase action again doubles number of identical DNA fragments
Stepped Art
Fig. 16-6, p. 256

22. Recording the Sequence
T C C A T G G A C C
T C C A T G G A C
T C C A T G G A
T C C A T G G
T C C A T G
T C C A T
T C C A
electrophoresis
gel
T C C
DNA is placed on gel
Fragments move off gel in size order; pass through laser beam
Color each fragment fluoresces is recorded on printout
T C
one of the many fragments of
DNA migrating
through the gel T
one of the DNA fragments
passing through a laser beam
after moving through the gel
T C C A T G G A C C A

23. Recording the Sequence
Fig. 16-8b, p.248

24. Genome Sequencing
Sequence of bacterium Haemophilus influenzae determined
Automated DNA sequencing now main method
Draft sequence of entire human genome determined in this way

25. Genome Sequencing
Fig a, p.250

26. Nucleotides for Sequencing
Standard nucleotides (A, T, C, G)‏
Modified versions of these nucleotides
Labeled so they fluoresce
Structurally different so that they stop DNA synthesis when they are added to a strand

27. Reaction Mixture Copies of DNA to be sequenced Primer DNA polymerase
Standard nucleotides
Modified nucleotides

28. DNA Fingerprints Unique array of DNA fragments
Inherited from parents in Mendelian fashion
Even full siblings can be distinguished from one another by this technique

29. Tandem Repeats
Short regions of DNA that differ substantially among people
Many sites in genome where tandem repeats occur
Each person carries a unique combination of repeat numbers

30. RFLPs Restriction fragment length polymorphisms
DNA from areas with tandem repeats is cut with restriction enzymes
Because of the variation in the amount of repeated DNA, the restriction fragments vary in size
Variation is detected by gel electrophoresis

31. Gel Electrophoresis DNA is placed at one end of a gel
A current is applied to the gel
DNA molecules are negatively charged and move toward positive end of gel
Smaller molecules move faster than larger ones
trophoresis.html

32. Gel Electrophoresis
Fig. 16-9b, p.249

33. Analyzing DNA Fingerprints
DNA is stained or made visible by use of a radioactive probe
Pattern of bands is used to:
Identify or rule out criminal suspects
Identify bodies
Determine paternity

34. Genomics
Structural genomics: actual mapping and sequencing of genomes of individuals
Comparative genomics: concerned with possible evolutionary relationships of groups of organisms

35. Reactions Proceed
Nucleotides are assembled to create complementary strands
When a modified nucleotide is included, synthesis stops
Result is millions of tagged copies of varying length

36. DNA Chips
Microarrays of thousands of gene sequences representing a large subset of an entire genome (p251)‏
Stamped onto a glass plate the size of a small business card (p251)‏

37. Genetic Engineering
Genes are isolated, modified, and inserted into an organism
Made possible by recombinant technology
Cut DNA up and recombine pieces
Amplify modified pieces

38. Engineered Proteins
Bacteria can be used to grow medically valuable proteins
Insulin, interferon, blood-clotting factors
Vaccines

39. Cleaning Up the Environment
Microorganisms normally break down organic wastes and cycle materials
Some can be engineered to break down pollutants or to take up larger amounts of harmful materials

40. Can Genetically Engineered Bacteria “Escape”?
Genetically engineered bacteria are designed so that they cannot survive outside lab
Genes are included that will be turned on in outside environment, triggering death

41. p.252

42. Engineered Plants Cotton plants that display resistance to herbicide
Aspen plants that produce less lignin and more cellulose
Tobacco plants that produce human proteins
Mustard plant cells that produce biodegradable plastic

43. Engineered Plants
Fig a, p.253

44. Engineered Plants
Fig b, p.253

45. The Ti plasmid
Researchers replace tumor- causing genes with beneficial genes
Plasmid transfers these genes to cultured plant cells
plant cell
foreign gene
in plasmid

46. The Ti plasmid
b The bacterium infects a plant and transfers the Ti plasmid into it.
a A bacterial cell contains a Ti plasmid (purple) that has a foreign gene (blue).
e Young plants with a fluorescent
gene product.
c The plant cell divides.
d Transgenic plants.
Fig , p.253

47. Genetic Changes
Humans have been changing the genetics of other species for thousands of years
Artificial selection of plants and animals
Natural processes also at work
Mutation, crossing over

48. First Engineered Mammals
Experimenters used mice with hormone deficiency that leads to dwarfism
Fertilized mouse eggs were injected with gene for rat growth hormone
Gene was integrated into mouse DNA
Engineered mice were 1-1/2 times larger than unmodified littermates

49. Transgenic Mice
Fig , p.254

50. Designer Cattle
Genetically identical cattle embryos can be grown in culture
Embryos can be genetically modified
create resistance to mad cow disease
engineer cattle to produce human serum albumin for medical use

51. Genetically Modified Animals
Fig a, p.254

52. Genetically Modified Animals
Fig b, p.254

53. Genetically Modified Animals
Fig c, p.254

54. Xenotransplantation
Researchers knockout the Ggta1genes in transgenic piglets
Ggta1 gene produces proteins that human antibodies recognize
Pig’s organs are less prone to rejection by a human

55. Safety Superpathogens
DNA from pathogenic or toxic organisms used in recombination experiments
Hok genes
NIH guidelines for DNA research

56. The Human Genome Initiative
Goal - Map the entire human genome
Initially thought by many to be a waste of resources
Process accelerated when Craig Ventner used bits of cDNAs as hooks to find genes
Sequencing was completed ahead of schedule in early 2001

57. Results of Gene Therapy
Modified cells alive in woman’s liver
Blood levels of LDLs down 20 percent
No evidence of atherosclerosis
Cholesterol levels remain high
Remains to be seen whether procedure will prolong her life

58. Using Human Genes
Even with gene in hand it is difficult to manipulate it to advantage
Viruses usually used to insert genes into cultured human cells but procedure has problems
Very difficult to get modified genes to work where they should

59. Ethical Issues
Who decides what should be “corrected” through genetic engineering?
Should animals be modified to provide organs for human transplants?
Should humans be cloned?