Genetic Engineering: PCR & Restriction Enzymes Technologies

1. DNA A Technology for Change

2. Polymerase Chain Reaction (PCR) 1. Amplifies (makes additional copies) of target DNA sequence

3. Polymerase Chain Reaction (PCR) Steps • CYCLE 1 • Denature target DNA sequence (heat). • Attach (anneal) 3’ and 5’ primers to opposite ends of • targeted sequence. • Use DNA polymerase to complete replication of target sequence (by producing complementary • sides of denatured strands). • CYCLE 2 • Repeat steps 1-3 above. • CYCLE 3…etc.

4. Restriction Enzymes • DNAase enzymes found in most cells • named for the source organism (e.g., • ECOR1, isolated from E. coli bacteria) • enzymes that “cut” DNA in specific • patterns “Blunt end” cutting type (Smal, Serratia marcescens) “Sticky end” cutting type (EcoR1, Escherichia coli)

5. Restriction Enzyme Examples

6. Gel Electrophoresis = Process for Separation of “cut” DNA piece Mixtures

7. Electrophoresis Gel Data Father Child Mother

8. Plasmids = transferable genetic elements, also called “replicons”

10. E. Coli “conjugation” – gene/plasmid transfer