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 Sometimes re-ligation conferred resistance w/out target sequence pUC plasmid series introduced blue/white screening using multiple restriction sites in lacZ 1975 plasmids commercially produced but majority still produced in labs

METHOD Isolation of DNA fragment Ligation into vector Transformation into host cell Select for cells with incorporated vector

TYPES OF CLONING TAQ 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 LIC Ligation independent cloning, 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 USER Urasil-specific Excision Reaction 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. cloning