Aulani "GE" Presentation 6 Recombinant DNA Technology Aulanni’am Biochemistry Laboratory Chemistry Department Brawijaya University

Aulani "GE" Presentation 6 The Mechanics of Biotechnology FBiotechnology was founded on the discovery of how to manipulate DNA outside the cell (in vitro) FIn particular, how to cut and splice different segments of DNA (genetic recombination) and how to move DNA from one organism to another (transformation) FBacteria can do this naturally

Aulani "GE" Presentation 6 Genetic Recombination in Nature FConjugation FTransformation FHomologous recombination FTransduction FTransposable elements

Aulani "GE" Presentation 6 Mechanics of Cloning DNA FWhat are the molecular tools required to clone a segment of DNA? FCloning = removal (excision) of a segment of DNA (usually a gene) from one genome and insertion of that segment into a vector, thereby creating a recombinant DNA molecule that can be inserted into and replicate in a host cell.

Aulani "GE" Presentation 6 Cloning DNA

Aulani "GE" Presentation 6 Mechanics of Cloning DNA F FHow to identify the segment of DNA to be cloned? F FHow to cut out (excise) the segment? F FWhat are the essential properties of a vector? F FHow to insert a DNA segment into a vector?   Proof of insertion?

Aulani "GE" Presentation 6 Restriction Nucleases (Endonucleases) F FRestriction nucleases cut DNA at specific sites by hydrolysis of phosphodiester bonds F FFour to eight (usually six) nucleotide sequences are recognized by the nucleases F FThe nucleases can cut straight across doubled stranded DNA to produce blunt or staggered ends.

Aulani "GE" Presentation 6 Restriction Nucleases

Aulani "GE" Presentation 6 Restriction Nucleases

Aulani "GE" Presentation 6 DNA Ligase catalyzes phosphodiester bond formation

Aulani "GE" Presentation 6 Vector Properties FOrigin of replication: Must be able to replicate in a host cell FCloning Site: Must have a restriction nuclease site for cloning, usually a unique site (occurs only once in the vector) F Selectable Marker: Must have a mechanism to select cells that contain the vector (usually antibiotic resistance)

Aulani "GE" Presentation 6 A Cloning Example FSource DNA is cut with a specific restriction enzyme and isolated (insert) FA cloning vector is cut with the same restriction enzyme FInsert and vector are mixed and DNA ligase is added FWhat are the possible outcomes of this reaction?

Aulani "GE" Presentation 6 Selection F FThe reaction mixture is added to bacterial cells made competent F FMost bacterial cells do not acquire plasmid F FPlasmid DNA expresses a gene for antibiotic resistance F FAntibiotics are added to the growth medium F FOnly cells that received a plasmid will grow

Aulani "GE" Presentation 6 Screening Candidates F FA number of antibiotic resistant colonies are usually selected (candidates) F FThe candidates are then screened to determine presence of the insert and the relative orientation. F FHow to eliminate candidates with no insert (religated vector)?

Aulani "GE" Presentation 6 DNA Restriction Mapping F FA physical map of a segment of DNA can be created by locating various restriction sites as landmarks F FInsertion of DNA into this segment will change the map relative to the site of insertion and can be used to “map” the insert

Aulani "GE" Presentation 6 Gel Electrophoresis

Aulani "GE" Presentation 6 Gel Electrophoresis Separation technique based on fragment size Fragments separated by electrical potential difference Incredible technique