1. DNA Technology and Genetic Engineering
Human protein
production
Transgenic
organisms
Forensic analysis
Human genome
project
RFLP analysis
DNA Technology and Genetic Engineering

2. Some uses of DNA Technology and Genetic Engineering
1: Production of human proteins
2: To identify people
3: To identify human diseases
4: To identify all human genes
5: To genetically engineer food
Supercoils
Coils
Nucleosomes
Histones
Nucleus
Chromosome
DNA
Cell

3. Use 1: To produce human proteins in bacteria (E. coli)
Example: Curing Pituitary Dwarfism

4. Early attempts to treat Dwarfism
The pituitary gland produces a protein that stimulates growth
People with certain types of dwarfism produce little
or no growth hormone
Researchers studied families with dwarfism and found that people with dwarfism have defective copies of the gene, GH1
Dwarfisms is a recessive disease that causes adults to be no more than 4 feet tall
Attempts to inject growth hormone from pigs (a strategy that worked previously for insulin) did not work – only GH from humans would work (until 1980’s source from human cadavers and up to 20,000 pituitaries were needed!)
Some of these pituitaries were contaminated with prions, which cause degenerative brain disorders

5. Solution: Use recombinant DNA to produce GH in bacteria
Recombinant DNA = DNA that results from combining DNA from different sources (ex. mouse + human DNA, human + bacterial DNA)
How make recombinant DNA? We need:
1) To isolate the gene of interest (human growth hormone) with restriction enzymes
2) A vector
3) To combine the gene of interest and the vector
4) Transfer the recombinant DNA to the host

6. 1) Isolate the gene of interest with restriction enzymes (DNA scissors)
Occur naturally in bacteria – why?
Hundreds are purified and available commercially
Recognize and cut at specific base sequences in DNA (usually 4-8 bases long)

7. Products generated by restriction enzymes
1) Sticky-end cutters
Enzyme Recognition site DNA after cuts
2) Blunt-end cutters
5’...GAATTC...3’
3’...CTTAAG...5’
5’...G
3’...CTTAA
AATTC...3’
G...5’
EcoRI 
5’...CCCGGG...3’
3’...GGGCCC...5’
5’...CCC
3’...GGG
GGG...3’
CCC...5’
SmaI 

8. Unit 4, Topic 5 - Genetic Engineering

9. Unit 4, Topic 5 - Genetic Engineering

10. 2) Vectors
Vector = something to carry the gene of interest into the host (i.e. bacteria)
A. Mechanical – micropipettes or gene guns
B. Biological – virus or plasmid
Plasmid –ring of DNA found only in bacteria
Cut both the plasmid and the gene of interest with the same restriction enzyme and their ends will stick together

11. 3) Combine the gene of interest and the vector

12. 3) Combine the gene of interest and the vector – a different picture

13. Restriction enzymes linearize a circular plasmid

14. More restriction enzyme action

15. 4) Transfer the recombinant DNA to the host
When the host cell copies its DNA, it also makes a copy of the plasmid, so we have lots of copies
Bacteria reproduce very quickly and have all the protein synthesis machinery (enzymes, ribosomes)
Insulin
Blood factor VIII-hemophilia
Antigens for vaccines
Cutting chromosomes in order to study individual pieces

16. Host cells produce protein products – ex. GH and insulin

17. Use 2: To identify people
Example: Forensic analysis (DNA fingerprinting)

18. RFLP Analysis RFLP = Restriction Fragment Length Polymorphism
Cut the same section of DNA with the same restriction enzymes
Two people will end up with different fragment sizes because their DNA is different

19. How can we see our DNA pieces after they are cut up?
Gel Electrophoresis – separates DNA fragments by size using electricity
DNA samples dyed and put into “lanes”
Pieces of chopped DNA move through jello-like gel
Bigger pieces move slowly, stay at top of gel
Smaller pieces move quickly, reach bottom of gel

20. Review: What is DNA made of?
DNA nucleotides:
Deoxyribose sugar
Nitrogenous base
Phosphate 3 2 1
Does DNA have a charge?
YES!
NEGATIVELY CHARGED

21. Gel electrophoresis
Gel electrophoresis and Southern Blotting

22. 1 2 3 4 5 Which suspect is guilty? DNA Fingerprint 1. Victim
3. Blood on suspect 1
4. Blood on suspect 2
5. Suspect 2
Which suspect
is guilty?

23. Is the defendant innocent or guilty?

24. Crime Scene ID

25. DNA
Fingerprinting
FBI probes 13 regions in the genome
Approximate probability that two people will match at a random DNA sample in one site is 1/10
The probability that two people will match at three sites is: 1/10 x 1/10 x 1/10 = 1/1000
Applying this probability to all 13 sites is one in ten trillion (or virtually 0)!

26. Paternity tests – Who is the father?

27. Use 3: To identify human genetic diseases
Screening for sickle-cell anemia

28. A single nucleotide change can make a difference
Wild-type allele
GAATTC
CTTAAC 
Restriction site
Mutant allele
GACTTC
CTGAAC ? 
Not a restriction site

29. Example: Sickle-cell allele destroys an MstII site

30. Southern blot

31. RFLP mapping of sickle-cell anemia in a family

32. Use 4: To find and identify all human genes
The Human Genome Project

33. Human genome facts
Started in 1990 and projected to take 15 years to finish. Working draft in 2000 and declared complete in 2003.
Goal to sequence all of our DNA, which is over 3 billion nucleotides!
Originally we thought we had over 100,000 genes, but we actually have ~20,000 genes

35. Use 5: Change characteristics
Transgenic organisms

36. These transgenic sheep carry a gene for a human blood protein, which they secrete in their milk. This protein inhibits an enzyme that contributes to lung damage in patients with cystic fibrosis and some other chronic respiratory diseases.

37. Transgenic Organisms Green-glowing aquarium fish (jellyfish genes)
Unit 4 - Code of Life
Transgenic Organisms
Green-glowing aquarium fish (jellyfish genes)

38. Transgenic Organisms Fast-growing fish (Salmon with Pout genes)

39. Transgenic Crops Herbicide-resistant crops (Round-Up Ready)
Molecular basis for the herbicide resistance of Roundup Ready crops.Todd Funke�, Huijong Han�, Martha L. Healy-Fried�, Markus Fischer�, and Ernst Schbrunn�,ｧ+Author Affiliations.�Department of Medicinal Chemistry, University of Kansas, Lawrence, KS 66045; and.�Department of Organic Chemistry and Biochemistry, Technical University Munich, D Garching, Germany.Edited by Brian W. Matthews, University of Oregon, Eugene, OR, and approved July 12, 2006 (received for review May 3, 2006) Next SectionAbstractThe engineering of transgenic crops resistant to the broad-spectrum herbicide glyphosate has greatly improved agricultural efficiency worldwide. Glyphosate-based herbicides, such as Roundup, target the shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, the functionality of which is absolutely required for the survival of plants. Roundup Ready plants carry the gene coding for a glyphosate-insensitive form of this enzyme, obtained from Agrobacterium sp. strain CP4. Once incorporated into the plant genome, the gene product, CP4 EPSP synthase, confers crop resistance to glyphosate. Although widely used, the molecular basis for this glyphosate-resistance has remained obscure. We generated a synthetic gene coding for CP4 EPSP synthase and characterized the enzyme using kinetics and crystallography. The CP4 enzyme has unexpected kinetic and structural properties that render it unique among the known EPSP synthases. Glyphosate binds to the CP4 EPSP synthase in a condensed, noninhibitory conformation. Glyphosate sensitivity can be restored through a single-site mutation in the active site (Ala-100萌ly), allowing glyphosate to bind in its extended, inhibitory conformation.

40. Unit 4 - Code of Life
Transgenic Crops
Pest-resistant crops (Bt toxin)

41. Golden rice
Transgenic rice produces beta carotene (we use it to make vitamin A) that gives the rice their golden color and their increased nutritional value. Vitamin A deficiency leads to vision problems and susceptibility to disease

42. genes into mutant cells
Gene Therapy
To insert normal
genes into mutant cells

43. Summary of the applications of DNA technology
Medicine - bacteria produce human proteins
To identify people
To diagnose genetic diseases
To find and identify all of the human genes
Gene therapy
Genetically engineered foods
Agriculture – bacteria that decompose nitrogen faster, disease and pest resistant plants, bigger, sweeter, more nutritious plants, bigger, leaner animals
Industry – bacteria used to break down pollutants