Circular RE map

Logic of constructing a circular restriction map

    Digestion of a linear DNA molecule with a restriction endonuclease (RE) that makes a single cut produces two fragments. In contrast, digestion of a circular DNA molecule with any RE that makes a single cut produces a single linearized fragment, all of which are necessarily of identical size. In the example, A & B each produce a linearized circular molecule, length 17kb [far left]. Inspection of the AB double digest data shows that the A & B sites are 5kb apart.  The unique map of A & B is thus easily drawn [left, middle].

    C cuts the circle twice, producing two fragments: there is only one possible map [middle, top]. Inspection of the AC double-digest data shows that A cuts the (smaller) 7kb C fragment.  The unique map of A & C is easily drawn
[top, right] .

    Inspection of the BC double-digest data [lower middle] shows that B cuts the (larger) 10kb fragment of C.  There is only one way to diagram this, and the map of B & C is easily drawn
[middle, bottom] .

    To integrate the AC and BC maps, recall that AB produces a 5kb fragment. Thus the B site is 5kb away from A. If B were clockwise from A, B would cut the smaller C fragment into 1kb and 6 kb in the BC diges], which the data show is not so.  If B is counter-clockwise from A, it would cut the larger C fragment into 4kb and 6kb fragments, which matches the data [right, bottom]. 

    The final map of ABC places A and B on either side of the C site, and all distances add up to 17kb, as expected [far right].

    Circular restriction maps are important in mapping plasmid DNA and mitochondrial DNA molecules. The backbone of an mtDNA map is frequently the placement of single-cut site REs, as above, A second clue in mtDNA from vertebrate animals is the occurrence of two Sst II sites at an interval of 1.6Kb between two highly conserved regions of the larger and smaller rRNA genes. These can serve to orient the restriction map to the functional map of the molecule.

All text material © 2012 by Steven M. Carr