分子生物学英文课件：genetic engineering.ppt

Ligation: Joining the target DNA to the vector DNA covalently using DNA ligase to form a recombinant DNA There are 4 kinds of ends ligated: Ligation between compatible sticky ends Ligation between blunt ends Ligation between homopolymer tails 4. Ligation between sticky ends with synthetic linker containing a convenient restriction enzyme sequence. AATTCxxxxxxA GxxxxxxTTCGA G AGCTT CTTAA A GAATTCxxxxxxAAGCTTCTTAAGxxxxxxTTCGAA ligase *Using the same one enzyme to cut *Ligated easily and conveniently *Self-reconnection of vector *Inserted in two directions 1) Sticky-end ligation *Using the same two enzymes to cut *Ligated easily and conveniently *no self-reconnection of vector *Inserted in one direction EcoRI HindIII EcoRI HindIII EcoRI EcoRI 2) Blunt-end ligation Recombinant DNA *to cut the DNA using the same or different enzymes that produce blunt ends or sticky ends that are trimmed to blunt ends by exonuclease. *more difficult ligation *2-direction insertion *self-reconnection *no easy way of retrieving the insert T4 DNA ligase vector *To cut the DNA using the same or different restriction enzymes that produce blunt or sticky ends that can be trimed to blunt ends. *To add a homopolymer tail to 3’end using terminal transferase *Increasing ligation efficiency *Decreasing self-reconnction *2-direction insertion *no easy way of retrieving the insert 3) Ligation between homopolymer tails 4) Ligation between sticky ends with synthetic linker GAATTCGGCTTAAGCC GGGAATTCCCCTTAAG EcoRI Cut with RE Ligase + linker *Cut target DNA using proper RE *Ligating blunt-end with a linker *Cut the vector and DNA with a linker using the same restriction enzyme and ligate them. Step 4. Introduction of recombinant plasmid into a bacterial cell to form recombinant bacterium Transformation: the process that plasmids can be introduced into bacterial cells. Transfection: Infection of a cell with purifi