Molecular Cloning

Definitions Cloning : Sub-cloning: Plasmid recombinant: Obtaining a piece of DNA from its original source (Genome) and introducing it in a DNA vector Sub-cloning: Transfer a cloned DNA insert or a part thereof from one vector to another vector Plasmid recombinant: Vector into which foreign DNA was introduced Recombinant organism Organism with a recombinant vector

Why clone? Separate, identify, manipulate or express a specific DNA fragment 3

Step 1- Separate Two approaches: Fragment/digest genomic DNA Generates a vast number of fragments May be difficult to find fragment of interest PCR Amplification Much less fragments Much easier to find sequence of interest

Cloning Generate compatible ends DNA ligation Restriction Enzyme Appropriate vector Fragment of interest DNA Recombination Intermolecular ligation Recombinant Intramolecular ligation Non-Recombinant Transformation Host Cells Recombinant cell Non-Recombinant cell 1 plasmid/1 cell

Amplification of Recombinant Plasmids Duplication 1 colony= 1 clone with 1 plasmid + 1 insert Bacterial growth

Screening or Identification of Recombinant Plasmid clones Restriction mapping Hybridization PCR

Expression Why? Produce the protein in a heterologous system Produce large quantities of the protein Purify the protein Study the protein’s activity

Amplification, Cloning & Expression of LacZ– Project II Part I PCR amplification & mutagenesis of LacZ Part II Cloning of LacZ in pUC19 Part III Enzyme assays of recombinant proteins

Purification & Digestion of LacZ Amplicon Purification by QiaQuick Removes buffer, salts, etc. Removes primers Removes enzyme Digestion Restriction sites NdeI and PstI were added to amplicon sequence Not present in LacZ sequence Unique in pUC19 vector Allows directional cloning