2. 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.jpghttp://w3.dwm.ks.edu.tw/bio/activelearner/14/images/ch14summary.gif

3. 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 Hydrogen bond Image taken without permission from http://www.bioteach.ubc.ca/MolecularBiology/RestrictionEndonucleases/endonuclease%202.gif Covalent bond

4. 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

5. Examples of Restriction Enzymes EnzymeOrganism sourceRecognized Sequence EcoRIEscherichia coli5' GAATTC 3’ 3' CTTAAG 5’ TaqIThermus aquaticus5' TCGA 3’ 3' AGCT 5’ HindIIIHaemophilus influenzae5'AAGCTT 3’ 3'TTCGAA 5’ BamHIBacillus amyloliquefaciens5' GGATCC 3’ 3' CCTAGG 5’ AluIArthrobacter luteus5' AGCT 3’ 3' TCGA 5’

6. 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’ Sticky ends

7. 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

8. Recombinant Plasmids 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

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

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

11. 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