Biotechnology: Tools and Techniques of the Trade

1. Biotechnology: Tools and Techniques of the Trade What can you do with DNA? Chapter 8

2. Success Criteria • At the end of class today, you will be able to: • Explain what a restriction enzyme is and what role they play in the cell • Explain how restriction enzymes have been used by biologists as a tool in the manipulation of DNA

3. Biotechnology biotechnology – manipulation of _______________ To study the functions of individual genes, molecular biologists will cut them out of a genome and place them into bacteria.

4. Basic Techniques Six basic techniques will be covered: • DNA isolation • restriction enzyme digest • PCR • gel electrophoresis • sequencing • gene cloning

5. 1. DNA Isolation

6. Isolating DNA Before DNA can be manipulated, it needs to be isolated from the cells. • Cell membranes are: • DNA precipitation • DNA isolation / storage

7. DNA Isolation

8. 2. Restriction Enzyme Digest pp 366 - 367

9. Restriction Enzymes

10. Restriction Enzymes restriction endonucleases – recognition site –

11. Restriction Enzymes Why are these enzymes used? Where do they come from? What role do they have in nature?

12. Restriction Enzymes Recognition sites. Palindromic. Which of the following would be palindromic? a) ATACTC b) AACGTACGTT c) CGGCCGGC d) ACGCGT

13. Restriction Enzymes Restriction enzymes are named according to the organism from which it was identified. Ex. EcoRI E - Escherichia co - coli R - strain RY13 I - 1st enzyme in this strain

14. Restriction Enzymes sticky ends – If the following piece of DNA is cut with Xho1, what ends would be created? (See page 367 for recognition site) 5’-ATTCGCTACTCGAGCTCGATCCTCGAGGCGCAATC-3’

15. Restriction Enzyme Cutting blunt ends –

16. DNA Ligase T4 DNA ligase –

17. Recombinant DNA recombinant DNA:

18. Recombinant DNA Animation