2. What is genetic engineering?
A direct, deliberate modification of an organism’s genome

3. So what does it look like?
A farmer mates his two largest pigs in hope of producing larger offspring. Unfortunately, he quite often ends up with small or unhealthy animals due to other genes that are transferred during mating. Genetic manipulation allows for the transfer of specific genes, so that only advantageous traits are selected.

4. So what does it look like?
Courts have, for thousands of years, relied on a description of a person’s phenotype (eye color, hair color, etc.) as a means of identification. By remembering that a phenotype is the product of a particular sequence of DNA, you can quickly see how looking at someone's DNA gives a clue to his or her identification.

5. So what does it look like?
Diseases are the result of a missing of dysfunctional protein, and we have generally treated the disease by replacing the protein as best we can, usually resulting on only temporary relief and limited success. Genetic engineering offers the promise the someday soon, fixing the underlying mutation responsible for the lack of a particular protein can treat these diseases far more successfully than we’ve been able to do in the past.

6. DNA Review
3 parts
5 C sugar, phosphate group, nitrogenous base

7. DNA review
Hydrogen bonds hold nitrogenous bases together

8. Cutting DNA
Typically, an enzyme (DNA helicase) unzips the two strands by breaking H-bonds
Can use heat instead

9. Cutting DNA
Other enzymes, called endonucleases, can cut DNA between sugar and phosphate
Called restriction enzymes

10. Cutting DNA Restriction Enzymes
Discovered by Drs. Arber, Smith and Nathans in 1950’s. Nobel Prize

11. Cutting DNA Bacteria naturally have these enzymes
Protect them from foreign viral DNA
Chews it up

12. Cutting DNA Often Palindromes!! Restriction enzymes are very specific
Will only cut at certain points
Often Palindromes!!

13. Naming restriction enzymes
1st letter of genus name, 1st 2 letters of species name, strain, and the # found in strain (I, IV)
TRY THESE:
Escherichia coli; strain R, 1st discovered
Haemophilus influenza; type d; 3rd discovered
Bacillus amyloliquefaciens; strain H; 1st discovered

14. Blunt v. sticky ends
Depending on how enzyme cuts, two types of ends are produced

15. The pieces
Each restriction enzyme cuts at a certain point, so pieces of DNA vary in size
Restriction Fragment Length Polymorphisms (RFLP)
Pieces can be sealed with DNA ligase

16. What other toys are there?
Reverse transcriptase
Isolated from HIV
Can make a piece of cDNA from an mRNA template

17. What other toys are there?
Gel electrophoresis
Used to analyze the pieces

18. Gel electrophoresis

19. Separation will depend on mass and charge
Shows the migration of a charged particle under the influence of an electric field
DNA is negatively charged so it will move towards the cathode (+)
Agarose acts as the molecular sieve. Made of agar and sugar. Contains small pores of different sizes.
DNA sample is treated with a loading dye so that you can see the movement of the DNA as it moves from – to + charges
Stained with ethidium bromide that binds with DNA. Use UV light to “light up” ethidium bromide. Problem here, ethidium bromide is carcinogenic so use caution!!

20. Putting it to practice Virtual Electrophoresis Lab
More Electrophoresis

21. Want to know exact size and sequence of DNA?
Size is calculated by the number of base pairs (bp)
Object
Size
Average E. coli gene
1300 bp
Entire E. coli genome
4,700,000 bp (4700 kb)
Human mitochondria DNA
16 kb
Epstein-Barr virus
172 kb
Human genome
3.1 billion bp

22. Want to know exact size and sequence of DNA
Sequence: want exact order of base pairs
Frederick Sanger
Sanger Method

23. Sanger Method

24. Sanger Method Isolate a fragment
Denature(with heat) to make a single template strand
Add
DNA polymerase
Regular nucleotides
Reaction-stopping nucleotides (ddATP, ddGTP, ddCTP, and ddTTP)
Reaction will stop when polymerase uses reaction-stopping nucleotides

25. Sanger Method
Put it all together (by hand or by machine) to get sequence

26. Polymerase Chain Reaction
Aka PCR
Artificial DNA replication
No culturing
Very sensitive
Can detect cancer from a SINGLE cell
Very fast and efficient

27. DNA Replication In Vivo (natural) In vitro (artificial)
RNA primase needed (makes primer for DNA polymerase)
DNA helicase to unzip DNA
DNA polymerase (from host organism)
Pre-made primers added (for DNA polymerase to use)
Heat used to unzip DNA
Taq polymerase from Thermus aquaticus (protein that can withstand heat)

28. PCR

29. PCR Steps Denaturation Priming Extension Repeat
Use heat (94C) to break H-bonds between strands
Priming
Cooled (50-65C) to allow primers to attach
Extension
Heated (72C) and allows for new strands to be made using Taq polymerase
Repeat

30. PCR Side notes Can get ONE MILLION copies of DNA within only 20 cycles
Can usually do cycles in 2-3 hours!
Concern: amplify “wrong” DNA (contamination)

31. DNA Fingerprinting
Chemical structure of everyone's DNA is the same. Only difference is the order of the base pairs
Every person could be identified by the sequence of their base pairs.
Examine a small number of DNA sequences that are known to vary among individuals.

32. Variable Number Tandem Repeats (VNTRs)
DNA has pieces that contain genetic information that codes for genes (exons) and pieces that, apparently, supply no relevant genetic information at all (introns).
Introns (junk genes) may have served some purpose in our evolutionary history
Introns may be 20 – 100 base pairs long

33. Your VNTRs are inherited from your parents
Shown below are the VNTR patterns for Mrs. Nguyen [blue], &
Mr. Nguyen [yellow]
Their four children:
D1 (the Nguyens' biological daughter)
D2 (Mr. Nguyen's step-daughter, child of Mrs. Nguyen and her former husband [red])
S1 (the Nguyens' biological son)
S2 (the Nguyens' adopted son, not biologically related [his parents are light and dark green]).

34. Applications of DNA Fingerprinting
Paternity and Maternity
Criminal Identification and Forensics
Personal Identification – your own personal bar code!

35. Putting it all together
By using all of the toys and procedures previously listed, we can now sufficiently take advantage of recombinant DNA technology

36. Recombinant DNA technology
Remove genetic material from one organism and combine it with the genetic material of a different organism

37. Recombinant DNA technology
Bacteria naturally do this
So we put them to work! 
Bacteria can be engineered to mass-produce substances such as
Hormones
Enzymes
Vaccines

39. Recombinant DNA technology
Want genetic clones– exact same DNA
General steps
Remove desired gene
Put gene into vector (plasmid or virus)
Vector inserts DNA into cloning host (bacterium or yeast)
Host produces protein of interest

40. Cloning vectors Must be able to carry donor DNA
Must be accepted by cloning host
OPTION 1: Plasmid
Small
Well-understood
Easy to manipulate
Easy to put into host

41. Cloning vectors OPTION 2: Bacteriophage Virus that infects bacteria
Small
Very easy to put into host

42. Vector Characteristics
When choosing a vector, scientists consider the following
Origin of replication so it can be replicated
Must accept DNA of desired size
Virus < plasmid < BAC < YAC
Contain gene that confers drug resistance
So we know that the host picked it up

43. E. coli and S. cerevisiae are excellent hosts
Host Characteristics
Fast growth
Easy to culture
Nonpathogenic
Genome well-known
Can accept vectors
Make lots of proteins
Holds onto foreign gene(s) for several generations
E. coli and S. cerevisiae are excellent hosts

45. Biochemical products Disease: dwarfism (p 302) Previous treatment:
Issues with old:
New treatment:

46. Biochemical products Disease: diabetes (p 302) Previous treatment:
Issues with old:
New treatment:

47. Biochemical products Disease: hemophilia A (p 302) Previous treatment:
Issues with old:
New treatment:

48. Genetically Modified Organisms
Aka GMOs
1st GMO: Pseudomonas syringae
Had gene that allowed ice to form easily on plants 
Altered gene to now prevent ice formation 

49. GMOs Frostban Product that prevents ice on potatoes and strawberries
Never commercially sold
Activists feared its use and dug up the strawberries before they could be spray-tested

50. GMOs Flavr Savr Commercially available for tomatoes Not a big hit
Allowed them to ripen slowly
Not a big hit

51. GMOs
Bioremediation
Engineered bacteria to clean up oil spills and degrade toxins

52. GMOs Plants Agrobacterium tumefaciens
Bacteria that is good at transferring DNA
Makes galls (plant tumors)
Ti (tumor-inducing plasmid)

53. GMOs Animals Mice, pigs, sheep
Virus transfects fertilized egg / embryo
Animals secrete proteins in milk (Pharming)
Have eukaryotic genes (better)

54. GloFish

56. Bioethics
Field that relates biological issues to human conduct and moral judgment