1. (How to Mess with DNA for Fun and Profit)
Biotechnology
(How to Mess with DNA for Fun and Profit)

2. What is Recombinant DNA?
Recombinant DNA is what you get when you combine DNA from two different sources
Example:
Mouse + Human DNA
Human + Bacterial DNA
Viral + Bacterial DNA
Human + (other) Human DNA

3. Why Make Recombinant DNA?
Recombinant DNA technology may allow us to:
Cure or treat disease
Genetically modify our foods to increase flavor, yield, nutritional value or shelf life
Better understand human genetics
Clone cells or organs

4. Molecular Biology’s Best Friend: Bacteria
Why use bacteria?
They’re relatively simple organisms
They reproduce very quickly and asexually
It’s pretty easy to get DNA back into bacteria after you’ve changed it
We can mess around with their DNA and kill them (whoops) and no one gets mad.
Their daughter cells will be identical

5. Transgenic Organisms- contain genes from other species
Recombinant DNA Fun…
Transgenic Organisms- contain genes from other species

6. How can you get a mouse to glow in the dark?
Took out the gene that causes green fluorescence in jellyfish

7. Can do the same for plants and firefly luminescence
In 1986, isolated the gene for luciferase, an enzyme that allows fireflies to glow and inserted it into a tobacco plant. = plants glowed in the dark

8. Transgenic Mice
Mice inserted with human genes that make their immune system act similarly to humans
Easier to test effects of various diseases

9. Transgenic Livestock
Many livestock have copies of growth hormone genes
Grow faster and leaner meat

10. Transgenic Plants Corn Plants with vitamins
Genes to produce natural insecticide
Genes to resist weed-killing chemical
Plants with vitamins
Helping all people get essential nutrients

11. How to make recombinant DNA

12. Plasmids Small circular pieces of “extra” DNA found in bacteria
Contain special genes that are not related to basic life functions
(ex. Antibiotic resistance)
Can be transferred from one cell to another

13. Restriction Enzymes
Specialized protein that cuts DNA in a very specific place
Different REs cut at different places along the nucleotide sequence

14. Restriction Enzymes cont.
Each restriction enzyme recognizes a specific sequence of nucleotides = restriction site
Sticky Ends: where the enzyme cuts allows for joining with another piece of DNA cut with the same enzyme
EcoRI
Staggered cut yields two double stranded DNA fragments with single stranded ends called sticky ends
Sticky ends: single stranded overhangs are produced. Can form hydrogen bonds with complementary sequences.
EcoRI

15. What do you notice about these restriction sites?
Restriction sites = places where RE cut
Restriction enzymes are palindromes on opposite strands…read the same backwards and forwards
Each enzyme has two identical active sites that break the DNA backbone of both strands in a double helix.

16. Practice: How many DNA fragments do you make?
How many DNA fragments do you make when the following DNA strand is digested with BamHI?
5’ –GGCCGGATCCGGATGGCCCGATGGATCC- 3’
3’ –CCGGCCTAGGCCTACCGGGCTACCTAGG- 5’ 3

17. The way it works…
Any piece of DNA cut with a certain restriction enzyme will “stick” to any other piece cut with that same RE, even if they come from different sources.
Sticky ends form hydrogen-bonded base pairs with complementary single stranded stretches of DNA

18. So why do we care?

19. Insulin for Diabetics OLD WAY NEW WAY
Early preparations of insulin were purified quite crudely from pancreas tissue extracted from animals - either pigs or cattle. These are known as animal insulin or “natural insulins”
Human insulin is made biosynthetically…identical to insulin found in human pancreas and unlimited supply because you are not depending on cow and pig pancreas.

20. Step 1:
Isolate the human gene responsible for producing insulin and decide where you want to put it
In this scenario, we decide to put our human DNA into the plasmid of E.coli, a very common bacterium.

21. Step 2 & Step 3:
2. Get the bacterial (plasmid) DNA out of E.coli. 3. Cut your human DNA and bacterial DNA with the same restriction enzyme
The chosen enzyme cuts the plasmid in one place and cuts the human DNA (with many restriction sites) into many fragments
In making the cuts, the RE makes sticky ends on both the human DNA and plasmid

22. Step 4:
Mix the cut human DNA, which contains the insulin gene, with the cut bacterial DNA
They’ll stick together because they were cut with the same restriction enzyme.
The two fragments use DNA ligase to permanently stick together.
Union is temporary, but it can be made permanent by the pasting enzyme- DNA ligase…catalyzes the formation of covalent bonds between adjacent nucleotides

24. Step 5: Transformation
Get your new recombinant plasmid back into the bacteria
Bacteria will take in DNA that’s floating around near them= transformation

25. Gene for human growth hormone
Recombinant DNA
Gene for human growth hormone
DNA recombination
Human Cell
Sticky ends
DNA insertion
Bacterial Cell
Bacterial chromosome
Bacterial cell for containing gene for human growth hormone
Plasmid

26. Done! Now your E.coli will use its new DNA to make human insulin.
Gene Cloning: Because they reproduce so quickly, you’ll soon have thousands, millions or billions of human insulin making machines

27. You’ve got clean human insulin that can be packaged and given to diabetic patients

28. Gel Electrophoresis Analysis

29. Comparing cut up DNA How do we cut DNA?
Restriction enzymes
How do we compare DNA fragments?
separate fragments by size
How do we separate DNA fragments?
run it through a gelatin
agarose
made from algae
gel electrophoresis

30. “swimming through Jello”
Gel electrophoresis
DNA moves in an electrical field…
DNA is negatively charged
size of DNA fragment affects how far it travels
small pieces travel farther
large pieces travel slower & lag behind
DNA        – +
“swimming through Jello”

31. Gel Electrophoresis Agarose gel acts like a mesh
Smaller DNA molecules move through faster than large DNA molecules
Large molecules are more likely to get tangled up in the gel and therefore move slowly
The consistency of agarose can directly effect the movement of DNA
Thick vs. thin
Plinko

32. Gel Electrophoresis - + DNA & restriction enzyme wells gel
Uses: forensics, paternity, evolutionary relationships, medical diagnosis
wells
longer fragments
power
source
gel
shorter fragments +
completed gel

33. Review Diagram