1. DNA Synthesis Eukaryotes DNA unwinds at a replication origin Two replication forks form Bidirectional synthesis of DNA Much more DNA; complexed with proteins Eukaryotes have much more DNA Yeast 250-400 replicons Mammalian cells up to 25,000 Solution: Multiple replication origins Yeast 4X that of E. coli Drosophila 100X that of E. coli

2. DNA Synthesis in Eukaryotes Pre-initiation replication complex: ORC Cdt1 Cdc6 MCM

3. Enzyme FunctionProkaryotesEukaryotes Helicase Primase Polymerase Proofreading Sliding clamp Prevent ss closure DnaB DnaG Pol III  sliding clamp SSBPs MCM Primase s.u. of  -polymerase Pol  and  PCNA (Proliferating Cell Nuclear Antigen) RPA (Replication Protein A) Comparison of some replication components

4. A Problem and a Solution

5. Telomere Formation

6. A model for t loop formation

7. Reproductive cloning Biotechnology/Cloning

8. Reproductive cloning

9. Therapeutic cloning

10. http://www.rndsystems.com/dam_public/6515.jpg Therapeutic cloning

11. First Cloning Experiment Cohen et al., 1973 “Recombinant DNA”

12. Cloning Vectors: pBR322 plasmid Cloning into pBR322

13. RT-PCR and Cloning