Recombinant DNA technology Allison, Ch8: Recombinant DNA technology and molecular cloning
Historical perspective Labmda phage
Restriction modification system
Restriction enzymes
DNA ligase
Sticky end ligation Blunt end ligation Modified blunt end ligation
Modified blunt end ligation
Molecular cloning
Vectors Carry DNA molecules Independent replication (origin) Unique restriction sites (MCS) Selectable marker Easy to transfer and recover from host
Choice of vectors
Plasmid Nomenclature pBR322 (p-plasmid, BR – origin: Bolivar and Rodriquez, 322 identifier) pUC18 (p-plasmid, UC- university of california, 18 – identifier)
Cloning using plasmid vector
Transformation and screening Transformation (competent cells) Recombinant selection Blue-white screening
Constructing libraries Library Genomic library cDNA library
Probes Homologous Heterologous EST probes Labeling an detection methods Radioactive Enzyme conjugates Chromogenic Fluorescent
Radioactive labeling
Labeling methods
Library screening
Restriction mapping
Electrophoresis
RFLP
Southern hybridization
DNA sequencing
Automated sequencing