Transcript Cloning
Cloning Using Plasmid Vectors Vector = a molecule used as a vehicle to carry foreign DNA into a host cell Simplest vector = plasmid Features of Plasmids Size Functions encoded Structure Nomenclature: R-plasmids ColE1 Now standardised Why are plasmids suitable cloning vectors? Generally do not kill host cell Relatively easy to purify Can be made small Why not use naturally occurring plasmids? Too large No selectable markers Lack of unique recognition sequences for restriction enzymes Replication Plasmid = replicon Requires an origin of replication (oriV) What is required? Functions of the ori region Host range Narrow vs Broad Copy number Features of Plasmid Cloning Vectors Contain an oriV that allows for high copy number, may have narrow (pUC) or broad (R) host ranges Small – why is this an advantage? Selectable Genes Unique restriction sites May have additional features such as mob sites, RNA polymerase promoters, etc. pBR322 1973-1978 Bolivar and Rodriguez derivative 322 4.36 Kb; ~16 copies per cell Narrow host range Encodes resistance to ampicillin and to tetracycline How can we tell if plasmid contains DNA of interest? Insertional inactivation Use BamHI site in Tetr gene for cloning Transform Plate cells on? Amp – why not Tet? Confirm by replica-plating on? Tet – what do you expect to see? Early 1980s – pUC series Features of pUC Plasmids Small Very high copy number No insertional inactivation Blue-White Selection Vector contains first 146 aa of the bgalactosidase gene (lacZ) (a-peptide) MCS embedded within this region Blue-White Selection Continued Host cell encodes carboxy terminal portion of lacZ Neither host nor plasmid encodes for entire protein Together produce enzyme that can cleave Xgal to produce blue precipitate What if foreign DNA inserted into MCS? Foreign DNA will contain a termination codon in the same reading frame as the a-peptide No a-peptide therefore no bgalactosidase and no blue coloured colonies pGEM series Additional Features Origin of DNA replication from ss filamentous phage such as F1 or M13 Phagemid vector T7 and SP6 promoters www.promega.com Variations on the pGEM theme pGEMT vectors (Hengen, 1995) Make use of unique property of Taq Facilitates easy cloning of PCR products how? Limitations?