1. DNA Technology

2. The process of manipulating genes for practical purposes.
Genetic Engineering
The process of manipulating genes for practical purposes.
Because all DNA has the same basic structure, we can cut and paste genes from one organism into the chromosome of another organism.

3. Recombinant DNA Vector Gene of Interest
DNA made from two or more different organisms
(Connecting DNA from different sources)
Gene of Interest
The gene to be inserted
(Codes for the trait we want expressed)
Vector
The DNA into which we will insert the gene of interest.
*Plasmids make great vectors*

4. Small, self-replicating circles of DNA (Found commonly in bacteria)
Plasmids
Small, self-replicating circles of DNA (Found commonly in bacteria)
Readily take up new DNA.
Self-replicating
Easily transferable from one cell to another.
Let’s swap DNA!

5. Steps in Genetic Engineering
Step 1: Cut Gene of Interest & Vector
with restriction enzymes.
gene from other organism
transformed bacteria
recombinant plasmid
vector
plasmid
cut DNA +
glue DNA

6. How Do We Cut DNA? Restriction Enzymes
Enzymes that cut DNA in specific locations
“Blunt” ends
CANNOT be glued to another piece of DNA.
“Sticky” ends
CAN be glued to another piece of DNA.

7. Why do we need “Sticky” ends?
Sticky ends make it possible to
“glue cut ends together.
MUST cut Gene of interest & Vector plasmid with the SAME restriction enzyme!!
MUST have
Complimentary Sticky ends!

8. How do we “Glue” sticky ends together?
Step 2: Connect DNA fragments together
How do we “Glue” sticky ends together?
DNA Ligase
An enzyme used to connect sticky ends of DNA fragments

9. + How do we get the recombinant plasmid into a cell?
I feel totally transformed!
Look What I found!
Bacteria will pick up foreign DNA and make it their own.

10. How do we find the bacteria that have been transformed?
We engineer a plasmid that helps us find the bacteria that pick it up:
Insert the gene of interest in the middle of the LacZ gene thus destroying it.
No LacZ gene = No blue pigment
LacZ gene
Codes for a blue protein
Now culture the bacteria in a petri dish with ampicillin in it.
Antibiotic resistance genes

11. Which cells Have the Recombinant Plasmid?
Gene of interest
Original plasmid
Plasmid & Gene anneal
2 Plasmids anneal with one another
Gene of Interest anneals with itself
Die
Grow (Blue)
Grow (White)
Culture the bacteria in agar containing ampicillin

12. Screening for Bacteria that Contain the Recombinant Plasmid
Bacteria take up plasmid
Functional LacZ gene
Bacteria make blue color
Bacteria take up recombinant plasmid
Non-functional LacZ gene
Bacteria stay white color
Which colonies do we want?

13. How do we get more of these transformed bacteria?
Single Transformed Bacteria
Feed them and they will reproduce!
Binary Fission
Copy DNA and split = “Cloning”
Culture Bacteria
Harvest (purify) protein
Gene Cloning

14. Word Processing Cut, Paste, Copy, Find…
So, Transformation is like:
Word Processing Cut, Paste, Copy, Find…
Cut
Restriction enzymes
Paste
Ligase
Copy
Plasmid replication
Binary Fission
Bacterial Transformation
Change the meaning of the message. 14

15. Many Uses of Restriction Enzymes…
Now that we can cut DNA…
We can COMPARE it!
Why?
Forensics
Medical diagnostics
Paternity
Evolutionary relationships
and more… 15

16. Can we add those little marshmallows?
Comparing DNA
Cut all DNA with same restriction enzyme
* Restriction fragments
Separate fragments by size
Gel Electrophoresis
Process that uses an electrical current to separate DNA fragments by size
DNA jello??
Can we add those little marshmallows? 16

17. “Swimming through Jello”
Gel Electrophoresis
A method of separating DNA fragments by size using an electrical field.
DNA is negatively charged so in an electrical field it moves toward the positive side
Small pieces travel faster/farther than larger pieces.
DNA         – +
“Swimming through Jello” 17

18. Preparing a Gel

19. DNA & Restriction enzyme Restriction Fragments
Gel Electrophoresis:
DNA & Restriction enzyme
Restriction Fragments
negative -
Completed gel
wells
Longer fragments
power
source
gel
Shorter fragments
Who’s going to
win the race? +
Electrical current
positive 19

20. DNA Fingerprint
A pattern of dark bands on photographic film made when an individual’s DNA restriction fragments are separated by gel electrophoresis, probed, and then exposed to an X-ray film.
Because each individual’s DNA is unique, each person has a unique DNA fingerprint

21. DNA Fingerprint Uses: Forensics
Comparing DNA sample from crime scene with suspects & victim
Suspects
You’re Under
Arrest!
Crime scene sample S1 S2 S3 V –
DNA  + 21

22. DNA Fingerprint Uses: Paternity Cases
Who’s the father? –
Mom F1 F2
child
DNA  + 22

23. PCR Process used to repeatedly copy the same piece of DNA many times.
Helpful in magnifying DNA from:
Crime scenes
Fossil remains
PCR Animation

24. Human Genome Project 3.2 billion base pairs in the human genome
Only 1-1.5% codes for proteins
Only about 30,000 to 40,000 genes

25. Why would we want to do this?
Genetic Engineering
Why would we want to do this?
To Create Drugs & Vaccines:
Create organisms (Animals or bacteria) that produce human proteins
Sheep that secrete a human blood protein
in their milk that can be used to slow lung
damage in cystic fibrosis patients.
Bacteria that produce:
Factor VIII-protein that promotes blood
clotting to treat Hemophilia
Growth hormones
Production of human insulin by bacteria
To Grow Organs for transplantation:
Mice that grow a human ear which can be used for transplant

26. To create better agricultural products:
Genetic Engineering
Why would we want to do this?
To create better agricultural products:
Delayed ripening & resistance to spoilage
Anti-softening tomatoes
Protect crops from insects: BT corn
Corn produces a bacterial toxin that
kills corn borer (caterpillar pest of corn)
Herbicide resistance: Cotton
Bacterial gene resistant to weed-killing herbicides
Improve quality of food: Golden rice
Rice producing vitamin A improves nutritional value

27. Animal Cloning
Dolly
1st Cloned Animal 1996

28. Problems with Cloning Few survive Fatally oversized
Problems in development
Why does it Fail?
During early development, many genes are permanently turned off by the addition of methyl groups.