Lecture 22

Lecture 22 - Lecture21 Lecture22:RecombinantDNA Cloning

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Lecture 21 Lecture 22: Recombinant DNA Cloning involves separating a specific gene or DNA segment from a larger chromosome;  attaching it to a small molecule of carrier DNA, replicating modified DNA  Results in selective amplification of particular gene; requires o Cutting DNA @ precise location by restriction endonucleases  o Selecting small molecule of DNA capable of self-replication by vectors  like plasmids o Joining 2 DNA fragments by DNA ligase to form recombinant DNA o Moving recombinant DNA from test tube to host cell o Selecting/identifying host cells that contain recombinant DNA Making Recombinant DNA restriction endonucleases  recognize and cleave DNA at specific DNA sequences  to generate smaller fragments o recognize palindromic sequences o can make straggered cuts   leaving 2-4 nucleotides of 1 strand unpaired  at each end; “sticky ends” can base pair with each other or complementary  sticky ends o blunt ends   cleaved at opposing phosphodiester bonds leavning no  unpaired bases on the ends DNA fragment to be closed joined to cloning vector by  DNA ligase o Joined to a vector digested by the  same  restriction endonuclease  o Sticky ends facilitates ligation reaction; blunt ends less efficient  o Can create new DNA sequences by inserting synthetic DNA fragments  (linkers) between ends ligated  o Polylinkers:  inserted DNA fragments with multiple recognition sequences  for restriction endonucleases (good for inserting additional DNA by  cleavage and ligation) Recombinant vector introduced into a host cell which amplifies the fragment Cloning Vectors Plasmids     : circular DNA molecules replicate separately from host chromosome o Introduced through  transformation o Incubate in calcium chloride solution; shock of increased temperature  causes cells to take up plasmid NDNA o Electroportaion:  cells incubated subject high-voltage pulse causes  bacterial membrane to be permeable to large molecules o Only few cells take up plasmid RNA; so must select ones that do o Use plasmid that includes a gene that the hose requires for growth, e.g.  gene for resistance to antibiotic
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Lecture 21 o Only cells transformed by the recombinant plasmid can grow in the  presence of that antibiotic; e.g.  bPR322 Has origin of replication Contains 2 genes for antibiotic resistance ( tet R  and  amp R ) to allow  ID of cells that contain intact plasmid or recombinant version  pBR322 cleaved at the amp-resistant element by  PstI foreign DNA ligated to cleaved site; where successful, the  amp-resistant element is disrupted while tet resistance is  still intact E.Coli cells transformed, grown on plates containing tet to 
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This note was uploaded on 02/25/2010 for the course BIOL 2800 taught by Professor Salomon during the Spring '09 term at Brown.

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Lecture 22 - Lecture21 Lecture22:RecombinantDNA Cloning

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