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It is thought that, together with enzymes that methyl portions of native DNA, restriction enzymes protect cells from DNA of invading organisms cutting such DNA into pieces, thereby restricting its activity. In this experiment, using agrees gel electrophoresis, the number and relative positions of restriction sites for three restriction enzymes, Score, Hinkle and Pull, on the circular plasmid abrupt were mapped by determining the length (in base pairs) of the DNA fragments obtained when cutting the plasmid with each of the restriction enzymes separately and each combination thereof.
In agrees gel electrophoresis, a molecular sieve is created such that the distance traveled in the gel toward the anode by any DNA fragment (all of which carry negatively charged phosphate groups in the presence of a basic buffer) is inversely proportional to its molecular weight. Further, such distance traveled has a linear relationship with the log of such fragment’s molecular weight.
Since DNA consists solely of deconstructionists that differ only by their bases and each base pair has approximately the same molecular weight, the distance traveled in the gel toward the anode by any DNA fragment also has a linear relationship with the log f its length (in base pairs).
Specifically, the restriction sites were mapped as follows: (i) lambda DNA was cut using the restriction enzyme Handbill to form fragments of known base pair lengths which were separated by agrees gel electrophoresis; (ii) abrupt was digested in seven different ways using the combinations of restriction enzymes discussed above and the fragments from such digests were separated in the same electrophoresis; (iii) using the data from the lambda DNA fragments, a regression was run to determine the relationship between the log of the number of base pairs in fragment and the distance traveled towards the anode during he electrophoresis; (iv) the base pair length of the fragments from each digest was calculated using the relationship determined from the lambda DNA data; and (v) the length of the fragments produced by the different digests were analyzed to produce a map, as will be discussed below. IV. Results. 1. Photo. Attached as Exhibit A-I is a photograph of the results of the electrophoresis performed using our abrupt digests. The results show significant smearing, likely the result of inadequate time allowed for digestion of the DNA.