1. Restriction Enzymes

2. WHAT ARE RESTRICTION ENZYMES?
are molecular scissors, or BETTER YET, molecular scalpels, that are able to cut DNA
are also called “restriction endonucleases”

3. WHERE DO RE’S COME FROM? Bacteria
“Immune system” to protect against bacteriophages (bacteria-infecting viruses like Lambda).

4. THOUGHT QUESTION Bacteria are prokaryotes.
Prokaryotes do not have a nucleus.
Both DNA and RE’s are in cytoplasm.
Why isn’t bacterial DNA cut by RE’s?

5. Bacteria “methylate” their own DNA.
Methylase will add a CH3 (methyl group) to the restriction site.
RE can’t bind … no cutting.
EcoRI Methylase
EcoRI
EcoRI Cuts at GAATTC
EcoRI Methylase Adds CH3 at GAATTC

6. HOW DO RESTRICTION ENZYMES WORK?
Usually cut DNA at a “palindrome” such as GAATTC.
Palindrome – word or phrase when spelled backwords, spells the same word or phrase
Ex. BOB
MADAM I’M ADAM
A Toyota! Race fast, safe car. A Toyota 5’ 3’
GAATTC
| | | | | |
“Restriction site” or
“Recognition Sequence”
CTTAAG 3’ 5’

7. HOW DO RESTRICTION ENZYMES WORK?
RE’s cut DNA’s phosphodiester bonds and hydrogen bonds.

8. HOW DO RESTRICTION ENZYMES WORK?
RE’s generate two different types of “cuts”
Blunt ends
Cut straight through the middle of the palindrome
Sticky ends
Uneven cut through the DNA leaves “overhang” of unattached bases

9. HOW DO RESTRICTION ENZYMES WORK?
Blunt vs. Sticky…which is more useful to genetic engineers?
RECOMBINANT DNA = DNA from 2+ sources

10. HOW IS DNA PASTED TOGETHER?
Ligase – another enzyme which reconnects phosphodiester bonds.

11. In short…
Enzymes
BUILD or
BREAK
chemical
bonds…

12. Enzymes - a brief reminder
Are proteins
Usually end with “-ase”
Speed up chemical reactions (catalysts)
Have specific shapes that fit specific substrates
Lower the activation energy of a reaction
Can be used over, and over, and over again
Often require “cofactors” to work properly
metal ions - ex. iron, zinc, copper
organic molecules (COENZYMES) generally derived from vitamins

13. WHAT ARE RE’S USED FOR?
Genetic engineering – pasting together DNA from two different organisms.
Spider silk gene in goats
Glo-fish
GFP addition (to tag and track gene expression)
Golden Rice
Human Insulin

14. WHAT ELSE ARE RE’S USED FOR?
Forensics – DNA Fingerprinting for crime scene investigation and paternity testing.
Everyone’s DNA has a different sequence – even though only 0.1% different.
How frequently would EcoRI cut DNA?
46= once every 4096 bp
Lambda (48,514 bp) would expect about 12 EcoRI sites

15. HOW ARE RE’S NAMED? After bacteria which produces them. Genus Species
1st letter
Species
1st two letters
Strain
Order Isolated
EcoRI
Recognition Site
G^AATTC
Escherichia
coli
RY13 I

16. HOW ARE RE’S NAMED? After bacteria which produces them. Genus Species
Strain
Order Isolated
EcoRI
HindIII
BamHI
Escherichia
Haemophilus
Bacillus
coli
influenzae
amylo. R d H I
III I
Recognition Site
G^AATTC
A^AGCTT
G^GATGC

17. Quick review… 1. Monomer of DNA = 2. Three parts of the monomer =
3. A - T with ___ hydrogen bond
4. Which is bigger -- purines or pyrmidines?
5. What two bases are pyrimidines?
6. Charge on DNA =
7. DNA has a ____-wise and ____-hand twist.
8. Bond between sugars - phosphates =
9. Restriction enzymes are found only in…
10. Same forwards and backwards =
11. Bacteria add ____groups to their DNA to prevent cutting.
12. Overhangs of unpaired bases =
13. In HindIII, the “in” comes from the _________ of bacteria.
14. Enzymes usually end in _____.

18. 1. Monomer of DNA = Nucleotide
2. Three parts of the monomer = Sugar (deoxyribose), phosphate, base
3. A - T with 2 hydrogen bonds
4. Which is bigger -- purines or pyrmidines? Purines
5. What two bases are pyrimidines? Cytosine and thymine
6. Charge on DNA = negative
7. DNA has a Clock -wise and right -hand twist.
8. Bond between sugars - phosphates = phosphodiester
9. Restriction enzymes are found only in… bacteria
10. Same forwards and backwards = palindrome
11. Bacteria add CH3 (methyl) groups to their DNA to prevent cutting.
12. Overhangs of unpaired bases = sticky ends
13. In HindIII, the “in” comes from the species of bacteria.
14. Enzymes usually end in -ase.