1. Restriction Enzymes AP Biology Unit 2 Images obtained without permission from http://w3.dwm.ks.edu.tw/bio/activelearner/14/images/ch14summary.gif and http://www.bioteach.ubc.ca/MolecularBiology/RestrictionEndonucleases/endonuclease%202.gif and http://www.symmation.com/gallery/images/restriction-enzyme-ecorV-th.jpg

2. Hydrogen bond Covalent bond Restriction Enzymes: Molecular Scissors • Restriction enzymes (endonuleases) cut DNA at specific sequences • What kinds of bonds are broken when restriction enzymes cut? • Covalent bonds (within a single strand) • Hydrogen bonds (between strands) as a result of the strands coming apart Image taken without permission from http://www.bioteach.ubc.ca/MolecularBiology/RestrictionEndonucleases/endonuclease%202.gif

3. Origins of Restriction Enzymes • Naturally found in different types of bacteria • Bacteria use restriction enzymes to protect themselves from foreign DNA • Bacteria have mechanisms to protect themselves from the actions of their own restriction enzymes • Have been isolated and sold for use in lab work

4. Examples of Restriction Enzymes

5. Sticky ends Sticky Ends vs. Blunt Ends • When restriction enzymes cut, they produce either • Sticky ends (single stranded sections at the ends) • Blunt ends 5’ - - - G A A T T C - - - 3’ I I I I I I I 3’ - - - C T T A A G - - - 5’

6. Recombinant DNA • Recombinant DNA is constructed using restriction enzymes • Important: In order to join 2 pieces of DNA together they have to be cut by the same restriction enzyme • Why? • Otherwise, the sticky ends won’t match– DNA can’t bind together

7. Step 1: Gene of interest and plasmid are cut with the same restriction enzyme Step 2: mix together Step 3: Add DNA Ligase to seal DNA back together Recombinant Plasmids

8. Question… • What kinds of bonds does DNA Ligase form? • Covalent bonds (between nucleotides in a single strand of DNA)

9. Create recombinant plasmid Transform plasmid into E. coli Putting It All Together

10. Restriction Analysis • Using restriction enzymes to find out information about a piece of DNA • We can use restriction enzymes to find out • The size of a plasmid • If there are any restriction sites for a particular enzyme on a piece of DNA (ex. EcoRI) • How many restriction sites for a particular enzyme • Where the restriction sites are located