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11 Cards in this Set
- Front
- Back
making Gene cloning possible, need...
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-bacterial restriction enzymes
-cloning vectors -methods for manipulating and characterizing DNA's |
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plasmid vectors why
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-must be able to clone recombinant DNA to be useful
-Most frags of genomic DNA are replication incompetent and must be joined by suitable replicon-usually a derivative of a plasmid, virus or chormosome |
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restriction enzyme properties
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-Endonucleases(not exonucleases-from end)-they cleave DNA strands internally
-are palindromes-DNA reads same 5to3 as 3to5 (EcoRI-5-GAATTC-3 and 3-CTTAAG-5) -1500 RE have been purified from bact. -Isoschizomers-diff RE that recog. same DNA seq |
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Plasmid Vectors characteristics
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-(2-6000bp) small circular DNA
-in bacteria often assoc. w/ pathological phenotypes(antibiotic resistance) -Episomal-independant of and easily seperated from host genome -contain DNA "Ori" seq(origin of rep start site)-allows replication of plasmid DNA indep. of host genome (through Rolling Circle Model) -capacle of replications to several hundred copies-provides amplification of recombinant DNA |
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Recognition sites
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-BRE recognize unique DNA seq (4-8bp)
-cleave phosphate bonds within both DNA strands -leave 5' phosphate and 3'OH |
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Laboratory Plasmids
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-"designer plasmids"
-to be Useful must have: 1)at least one unique RE site(only once in plasmid) for insertion of foriegn gene. Many have multiple cloning sites MCS-region w/ several unique RE sites(allows wide use of plasmid). Unique RE sites must be in non-essential part of plasmid so you don't interupt plasmid func. 2)An Antibiotic reistance gene-so bact harboring plasmid will be antib. resistent and can be selected from those that don't 3)an origin of replication |
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Complementary overhangs
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-many RE cleave DNA leaving "sticky" comp. overhangs
-makes it easy to "stitch" together DNA w/ matching overhangs BLUNT ends-(DraI) hard to ligate-no overhang 5'OVERHANG-(EcoRI) 3' is bigger,5-G AATCC-3 and 3-CTTA G-5 3'OVERHANG-(Kpn1) 5' bigger, 5-GGTAC C-3 |
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Recombinant Plasmid
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created by:
1)inserting a gene of interest(which has overhangs corresponding to RE site) into plasmid that had been cut once in non-essential region 2)complementary overhangs are ligated w/ DNA ligase |
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Joining comp overhangs
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-allows joining of two diff DNA frags in predictable ways
-complementary overhangs joined by base pairing -2 diff DNA frags created w/ same RE can be annealed to create recombinant DNA-single DNA molec made from two or more diff DNA moecs. -This recreates RE site |
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RE cutting frequency
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-inversely proportional to recog seq length
-a 4-base cutter will cut DNA more frequently and into smaller peices than a 6-base cutter -allows determining of size dist. of frags from RE digestion -computers scan DNA seq to predict RE sites and make "restriction map" for DNA molec |
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RE bacterial defense
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-RE are part of bact defense against bacteriophage
-RE degrade entering foreign DNA -Bact protect own DNA w/ DNA methylases-these enzymes add CH3 to bact adenosine or cytosine.-this blocks RE cleavage Protection from-"modification-restriction" system |