Restriction Enzymes/plasmids

Front 1

making Gene cloning possible, need...

Back 1

-bacterial restriction enzymes
-cloning vectors
-methods for manipulating and characterizing DNA's

Front 2

plasmid vectors why

Back 2

-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

Front 3

restriction enzyme properties

Back 3

-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

Front 4

Plasmid Vectors characteristics

Back 4

-(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

Front 5

Recognition sites

Back 5

-BRE recognize unique DNA seq (4-8bp)
-cleave phosphate bonds within both DNA strands
-leave 5' phosphate and 3'OH

Front 6

Laboratory Plasmids

Back 6

-"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

Front 7

Complementary overhangs

Back 7

-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

Front 8

Recombinant Plasmid

Back 8

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

Front 9

Joining comp overhangs

Back 9

-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

Front 10

RE cutting frequency

Back 10

-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

Front 11

RE bacterial defense

Back 11

-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