1. Restriction Enzymes and their use in Biotechnology (Recombinant DNA and DNA fingerprinting)

2. Learning Targets
Explain what restriction enzymes are and how they are used in recombinant DNA technology and DNA gel electrophoresis.
Describe how DNA gel electrophoresis is used in the technology of DNA fingerprinting.
Explain how chemical features of DNA are utilized in gel electrophoresis and DNA extraction.

3. Restriction Enzymes Found in Bacteria and Archaea
Restriction enzymes cut invading DNA molecules at a limited number of specific DNA sequences, called restriction sites. (We have now discovered over 3,000 restriction enzymes).
Used as a way to protect themselves against viral DNA.

4. Using Restriction Enzymes to Make Recombinant DNA
The restriction sites are palindromes and a restriction enzyme will usually make many cuts in a DNA molecule yielding a set of restriction fragments.
The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that can bond with complementary “sticky ends” of other fragments.
Palindromes in DNA: The complementary sequence to a segment of DNA is the backwards version of its own sequence. For example,
the complementary strand to C-T-T-A-A-G is G-A-A-T-T-C.

5. Restriction Endonucleases
Examples of Type II Restriction Endonucleases
Blunt ends
Cohesive
“sticky” ends
Where do restriction enzymes get their names?

6. Restriction Endonucleases
Examples of Type II Restriction Endonucleases
An E. coli strain that carries the cloned Hpa I gene from Haemophilus parainfluenzae.
Escherichia coli
Haemophilus influenzae
An E. coli strain that carries the cloned Pst I gene from Providencia stuartii.
Where do restriction enzymes get their names?

7. Restriction Endonucleases used in recombinant DNA technology
A restriction enzyme cuts the DNA from two different organisms, leaving sticky ends exposed.
DNA ligase is used to seal the desired segments together.
Results in DNA from one organism combined with DNA from another organism (recombinant DNA)

8. Applications Genetically Modified Organisms
Gene that produces a unique trait (ex: herbicide resistance.
Soybean DNA with a nonessential gene.
Soybean plant now has resistance to the herbicide.

9. Another application: Forensic Evidence
Restriction enzymes are used to cut DNA and use for the process of DNA fingerprinting.
DNA “fingerprints” obtained by analysis of tissue or body fluids found at crime scenes can provide definitive evidence that a suspect is guilty or not.
DNA fingerprinting can also be used in establishing paternity.

10. A VERY simplified look at the process:
DNA discovered at crime scene.
Amplify DNA with Polymerase Chain Reaction.
Cut DNA with known restriction enzyme (e.g. EcoRI) to produce Restriction Fragment Length Polymorphisms (RFLP’s).
Analyze RFLP’s with gel electrophoresis.

11. Polymerase Chain Reaction
The PCR procedure is used to make multiple copies of a specific segment of DNA.

12. DNA Fingerprinting
Restriction fragment analysis can rapidly provide useful comparative information about DNA sequences.
Gel electrophoresis separates DNA restriction fragments of different lengths.

13. “Restriction Fragment Length Polymorphism”
DNA Fingerprinting
A DNA fingerprint is a specific pattern of bands of RFLP markers on a gel:
RFLP =
“Restriction Fragment Length Polymorphism”
DNA segments with EcoRI restriction sequences in the same place will produce RFLP’s of the same size and thus the same banding pattern.

14. There has been a terrible crime….

15. The victim, found bear-ly alive
The victim, found bear-ly alive. Was it a simple petting or a brutal assault? Or could it be that the case isn’t so black and white….

16. Possible Suspect: Rose
Rose has always been resentful of pandas. It started when a Dalmation ate her ice cream at the zoo. “NO!” she yelled, “BAD PANDA!” All of her friends laughed and nobody corrected her. To this day Rose thinks twice before calling the fire department, just in case they bring their pandas with them.

17. Possible Suspect: Bob
Bob is a bamboo floor salesman, a natural enemy of the panda. “I know they just eat it to be spiteful,” says Bob. “They clearly don’t know how to use it to make beautiful, long-lasting floors for a fraction of the cost of traditional hardwood like I do. I think that makes them jealous. Plus, I tried to eat it and it tastes terrible. No panda eats that for real.”

18. Possible Suspect: Jane
Jane loves pandas! She loves them so much that she has all kinds of panda accessories. She has a panda mug, a panda stuffed animal, a panda t-shirt, and a panda rug! “What’s my favorite kind of bear? Uhhhh PANDA! Duh! What’s my favorite kind of panda? That’s a little trickier…I’d say lightly braised with horseradish on a bed of greens.”

19. Possible Suspect: Ragrox, Destroyer of Worlds
Interviewer: So tell me Ragrox, what’s it like to get to know the ‘real’ you?
Ragrox: AAFAADHGUGUGssshduAGHGAGGGGHUUUUULTHKUTK!

20. The evidence left behind
A smattering of skin cells were found on the slain bear. Evidence left behind! The cells were collected and sent to the lab for processing. DNA was extracted from the cells and sent to the best scientists in the land…

21. Collect the evidence
Each person needs a sheet of DNA, four markers or colored pencils and a pair of scissors.
Take the DNA and cut it out. Connect it into one long strand of DNA with tape. This is the strand of DNA recovered from the scene.
Color code all of the restriction sites on the strand of DNA. Draw, BUT DO NOT CUT, where each of the cuts would be made by the restriction enzymes. Ex:

22. Gel electrophoresis of possible suspects DNA
Side of gel near positive electrode
Gel electrophoresis of RFLPs from DNA treated with HindIII
Side of gel near negative electrode
Rose
Bob
Jane
Ragrox, destroyer of worlds