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 replicons Mammalian cells up to 25,000 Solution: Multiple replication origins Yeast 4X that of E. coli Drosophila 100X that of E. coli

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

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

A Problem and a Solution

Telomere Formation

A model for t loop formation

Reproductive cloning Biotechnology/Cloning

Reproductive cloning

Therapeutic cloning

Therapeutic cloning

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

Cloning Vectors: pBR322 plasmid Cloning into pBR322

RT-PCR and Cloning