1. WHAT ARE RESTRICTION ENZYMES? Restriction Enzymes – molecular scissors able to cut DNA nuclease Enzymedigests nucleic acids

2. WHAT DNA SEQUENCES DO RESTRICTION ENZYMES CUT? Usually cut DNA at a “palindrome” such as GAATTC. GAATTC CTTAAG 5’ 3’ 5’ | | | “Restriction site” or “Recognition Sequence” 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

3. WHAT PARTS OF DNA ARE CUT? RE’s cut DNA’s phosphodiester bonds and hydrogen bonds.

4. WHAT’S LEFT AFTER RESTRICTION ENZYMES CUT DNA? - RE’s generate two different types of “cuts” - Blunt cuts. - Sticky ends

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

6. HOW ARE RE’S NAMED? After bacteria which produces them. GenusSpeciesStrain Order Isolated Escherichia coli R I EcoRI Haemophilus influenzae d III HindIII Bacillus amyloliquefaciens H I BamHI Recognition SiteG^AATTC A^AGCTTG^GATCC

7. WHAT MUST BE AVAILABLE FOR RESTRICTION ENZYMES TO WORK? All enzymes need the correct: Why are enzymes so needy? Need the same environment as the cell that produced them. 1.temperature 2. pH 3. salt concentration “buffer”

8. WHAT ARE RE’S USED FOR? Genetic engineering – pasting together DNA from two different organisms. Examples: Bacteria with human insulin gene Bacteria with human insulin gene Golden Rice with Vitamin A gene Golden Rice with Vitamin A gene Cows with human milk protein genes Cows with human milk protein genes Goats with spider silk genes Goats with spider silk genes

9. WHAT ARE RE’S USED FOR? Forensics (not always crime scenes) Forensics (not always crime scenes)

11. Examples of Transgenic Organisms “Transgenic” – moving genes across species Examples: Bacteria with human insulin gene Golden Rice with Vitamin A gene Cows with human milk protein genes Goats with spider silk genes Goats with spider silk genes

12. HOW DO RESTRICTION ENZYMES WORK? Which are more useful in genetic engineering? RE’s that generate sticky ends or ones that produce blunt cuts? STICKY ENDS

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