Topics to be covered Principles of ligation reactions Properties of DNA ligases Compatible ends Dephosphorylation of DNA using phosphatases Ligase independent ligation Introduction of DNA into cells –Transformation, conjugation, electroporation

DNA ligation DNA ligase can join matching sticky ends of DNA pieces from different sources that have been cut by the same restriction enzyme The mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3' ends of one nucleotide, ("acceptor") with the 5' phosphate end of another ("donor"). ATP is required for the ligase to work

Properties of commonly used ligases

„Similarity“ of ATP and NAD+

DNA ligation by E. coli or T4 DNA ligase Removal of terminal phosphate group prevents ligation Only sequence compatible ends can be ligated, but… Compatible ends can be joined Compatible ends can be joined but not recut

Maximizing formation of recombinant molecules

Ligase-independent cloning of PCR products

Transformation, Transduction, Conjugation Recombinant DNA molecules made in cloning should be introduced into suitable host organisms where they can replicate Introduction of recombinant DNA into host strains –Transformation: via natural or chemically induced competence for DNA uptake, bacteria, yeasts –Transduction: recombinant DNA is packaged into a phage particle and phage delivers DNA into target cell, bacteria –Conjugation: transfer of genetic material between two bacterial cells in direct contact via contacts made with pili, bacteria –Electroporation: cells shocked with an electric field of kV/cm which is thought to create holes in the cell membrane through which the plasmid DNA enters, bacteria, yeasts, mammalian cells –Viral transformation: recombinant DNA is packaged into a viral particle and virus delivers DNA into target cell, plants, mammalian cells

Introduction of the cloned DNA into a host organism by electroporation Transformation by electroporation is often used to get recombinant DNA into cells In electroporation, cells are mixed with DNA and exposed to brief high-voltage electrical pulses which makes the cell envelope permeable and allows entry of the DNA Electroporation is a quick process and works for most types of cells

Electroporation BioRad MiniPulser Electroporation System

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