1. Biotechnology I

2. POINT > Define what restriction enzymes are POINT > Describe how restriction enzymes cut DNA POINT > Show how restriction enzymes facilitate recombinant DNA technology POINT > Describe the basics of gel electrophoresis

3. POINT > Define restriction enzyme 1962: a “molecular scissors” was discovered in bacteria cells E. coli and other bacteria have an enzymatic immune system that recognizes and destroys foreign DNA (cuts it into pieces) 3,000 restriction enzymes have been identified, around 200 have unique properties, many are purified and available commercially Use of restriction enzymes has made cloning and other DNA technologies possible

4. POINT > Define restriction enzyme Restriction enzymes are named for the bacterial genus, species, strain, and type from which they were isolated Example: EcoR1 Genus: Escherichia Species: coli Strain: R Order discovered: 1

5. WB CHECK: What do restriction enzymes do? Where do restriction enzymes come from? Bacteria use restriction enzymes as a defense against what?

6. Restriction enzymes recognize specific DNA sequences (usually 5-6 base pairs long) and cut the DNA at those sites Cut sites are called restriction sites These cuts result in pieces of DNA called restriction fragments (usually a few hundred or thousand bases long) These DNA fragments can be separated by size using gel electrophoresis POINT > Describe how restriction enzymes cut DNA

7. Restriction sites usually have symmetry (palindromic) “Step on no pets” Bam H1 site: 5’-GGATCC-3’ 3’-CCTAGG-5’

8. POINT > Describe how restriction enzymes cut DNA Many restriction enzymes cut in a staggered fashion, which results in fragments with “sticky ends” EcoRI 5’…GAATTC…3’ 3’…CTTAAG…5’ Other restriction enzymes cut in a direct fashion, leaving “blunt ends” PvuII 5’…CAGCTG…3’ 3’…GTCGAC…5’

10. WB CHECK: How many base pairs long are most “restriction sites”? How long are the DNA sequences that result from being cut by restriction enzymes? What are the pieces of DNA that have been cut by restriction enzymes called? Restriction enzymes cut at specific places in DNA. What do we call these places?

11. POINT > Show how restriction enzymes facilitate recombinant DNA technology Recombinant DNA is used for: RFLP analysis (Restriction Fragment Length Polymorphism) and DNA fingerprinting DNA sequencing to compare individuals and species Recombinant DNA for genetically modified organisms and production of proteins like insulin in bacteria Large scale analysis – gene chips to screen for potential diseases

12. Human DNA cut with EcoRI Baboon DNA cut with EcoRI 5’-C-G-G-T-A-C-T-A-G 3’-G-C-C-A-T-G-A-T-C-T-T-A-A- A-A-T-T-C-A-G-C-T-A-C-G-3’ G-T-C-G-A-T-G-C-5’ + 5’-A-C-G-G-T-A-C-T-A-G A-A-T-T-C-A-G-C-T-A-C-G-3’ 3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A G-T-C-G-A-T-G-C-5’ Complementary base pairing + DNA Ligase Recombinant DNA molecule 5’-A-C-G-G-T-A-C-T-A-G-A-A-T-T-C-A-G-C-T-A-C-G-3’ 3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A-G-T-C-G-A-T-G-C-5’ POINT > Show how restriction enzymes facilitate recombinant DNA technology

13. 5’-C-G-G-T-A-C-T-A-G 3’-G-C-C-A-T-G-A-T-C-T-T-A-A- A-A-T-T-C-A-G-C-T-A-C-G-3’ G-T-C-G-A-T-G-C-5’ + 5’-A-C-G-G-T-A-C-T-A-G A-A-T-T-C-A-G-C-T-A-C-G-3’ 3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A G-T-C-G-A-T-G-C-5’ Complementary base pairing + DNA Ligase Recombinant DNA molecule 5’-A-C-G-G-T-A-C-T-A-G-A-A-T-T-C-A-G-C-T-A-C-G-3’ 3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A-G-T-C-G-A-T-G-C-5’ Any two pieces of DNA that are cut with the same restriction enzyme can be combined if they have sticky ends

14. WB CHECK: Which restriction enzyme shown here would be no good for recombinant DNA technology?

15. Gel electrophoresis is a technique that uses an electric current to separate DNA restriction fragments by size DNA is (-) charged and always moves to the (+) Smaller fragments move more quickly through the gel POINT > Describe the basics of gel electrophoresis

19. _ + DNA is negatively charged from the phosphate backbone Visualize DNA with ethidium bromide – fluoresces ONLY when bound to DNA POINT > Describe the basics of gel electrophoresis

21. WB CHECK: Why do DNA fragments move toward the positively charged end of an electrophoresis gel? Which DNA fragments move slowest? What are three ways scientists use DNA technology? If two restriction fragments were cut with the same restriction enzyme, and the sticky ends matched up, what enzyme is needed to finish putting them together? https://www.youtube.com/watch?v=aA5fyWJh5S0

22. Homework: Read your textbook pages 403-405 Workbook pages 247-250 Restriction enzyme W.S.