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MOLECULAR CLONING BY LIZ GLENN HISTORY • 1970 discovery of restriction endonucleases in bacteria • DNA ligase used to join sections of DNA, termed recombinant DNA • Ligase used to join fragments to bacteriophages or plasmids • First recombinant DNA molecule created in 1972 by Paul Berg HISTORY • First successful transformation in 1973 by Boyer, Cohen and Chang • Digested plasmid pSC101 with enzyme EcoRI which targets G/AATTC palindromic sequence • Ligated into another plasmid using restriction enzymes • Transformed into new strain of E. coli • Conferred tetracycline resistance HISTORY • • pBR322 first plasmid • pUC plasmid series introduced blue/white screening using multiple restriction sites in lacZ • 1975 plasmids commercially produced but majority still produced in labs Sometimes re-ligation conferred resistance w/out target sequence METHOD • Isolation of DNA fragment • Ligation into vector • Transformation into host cell • Select for cells with incorporated vector TYPES OF CLONING TAQ LIC USER • • • Ligation independent cloning, • Urasil-specific Excision Reaction Taq polymerase Isolated from Thermus aquiticus • Works at high temp • Adds single A on 3’ ends in PCR • Kits with vectors already linearized and “tailed” w/ T overhang no DNA ligase • T4 polymerase leaves ssDNA overhang > 12 nu. on target DNA • When mixed target and appropriate vector will anneal • Strong enough to be transformed • Errors fixed in vivo • Restriction enzyme and ligase independent • Taq to amplify target w/ single Urasil base • USER enzyme cleaves target at Urasil base forming overhang • First created in 1990’s APPLICATIONS • Gene expression • Production of recombinant proteins eg. Hepatitis B vaccine producing HBsAG, a viral envelope protein • Transgenic organisms eg. Glofish • Gene therapy- limited success REFERENCES • Cohen et al. “Construction of biologically functional bacterial plasmids in vitro”. • Hershfield et al. “Plasmid ColE1 as a molecular vehicle for cloning and amplification of DNA”. • Wilson et al. “Molecular cloning of fragments of bacteriophage T4 DNA”. • Aslanidis et al. “Ligase-independent cloning of PCR product”. • New England Biolabs Inc. website. www.neb.com/applications/cloning-and-synthetic-biology/usercloning
