1. SBI 4U December 2012 Manipulating & Cloning DNA

2. Introduction Insulin, diabetes and genetic engineering Genetic engineering: the intentional production of new genes and alteration of genomes by the substitution or introduction of new genetic material Bacteria and versatility  reproduce fast, inexpensive and contain plasmids

3. Introduction Recombinant DNA: a DNA strand that is created using DNA pieces from two or more sources. Ex: E.coli and biosynthetic insulin

4. Restriction Enzymes Restriction enzyme: an enzyme that cuts DNA at a specific location in a base sequence; also called restriction endonuclease; 2500 have been found! Each restriction enzyme recognizes a specific sequence of nucleotides on a DNA strand  recognition site When restriction enzyme cuts DNA molecule, the pieces are called restriction fragments

5. Restriction Enzymes Restriction enzymes cut only at one recognition site and in one direction Ex: EcoRI 1) Enzyme EcoRI bind to a recognition site with base pair 5’- GAATTC-3’ 2) EcoRI cuts bond phosphodiester bonds between this sequence and the DNA backbone 3) Another EcoRI makes the same cut in the complementary DNA strand 4) Result: complementary “sticky” ends

6. Restriction Enzymes 2 possible outcomes: blunt ends and sticky ends Blunt end: the end that remains after restriction enzymes cut straight across a DNA strand Sticky end: the end that remains after restriction enzymes cut on a zigzag across a DNA strand

7. Restriction Enzymes What is the function of restriction enzymes? In essence, their function is to protect the cells in which they are found. Ex: viral DNA/RNA injected into bacterial cell

8. DNA Ligase DNA Ligase: enzyme that is used to join cut strands of DNA Works best with sticky ends 2 nd form  T4 DNA ligase – works well with blunt ends

9. Plasmids Plasmids: small, circular loops of DNA found in bacteria Replicate independently Mutations of plasmids A cell that is able to take up foreign DNA is called a competent cell

10. Plasmids A plasmid designed to transfer foreign genetic material into a cell is called a vector The copy number is variable  what happens if the copy number is high? Ex: insulin