Recombinant DNA technology

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Recombinant DNA Technology
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Presentation transcript:

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

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