Restriction Enzyme Mapping of pBR322 Using Agarose Gel Electrophoresis.
Section: Thursday, 1:10 pm
Date of Experiment: October 25, 2012
Restriction enzymes (or restriction endonucleases), originally isolated from Haemophilus influenzae in 1970, are enzymes within a cell that cleave foreign DNA within a specific and predictable nucleotide sequence (known as a restriction site) regardless of the source of such DNA. Such restriction sites generally are four to eight base pairs in length. It is thought that, together with enzymes that methylate 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 agarose gel electrophoresis, the number and relative positions of restriction sites for three restriction enzymes, EcoR1, HincII and PvuII, on the circular plasmid pBR322 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 agarose 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 deoxyribonucleotides 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 of its length (in base pairs).
Specifically, the restriction sites were mapped as follows: (i) lambda DNA was cut using the restriction enzyme HindIII to form fragments of known base pair lengths which were separated by agarose gel electrophoresis; (ii) pBR322 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 the 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.
1. Photo. Attached as Exhibit A-1 is a photograph of the results of the electrophoresis performed using our pBR322 digests. The results show significant smearing, likely the result of inadequate time allowed for digestion of the DNA. The smearing is particularly troublesome in the lambda DNA digestion lane, where it prevents the creation of the standard curve necessary for analysis of the pBR322 digests. Consequently, a photograph of the results of the “class” electrophoresis (attached as Exhibit A-2) was used in the analysis/discussion below.
2. Table 1. The following table presents the data obtained by performing electrophoresis on the lambda DNA fragments obtained by cleaving such DNA with HindIII.
|Band # |( Fragment Length (Base Pairs (bps)) |Log[( Fragment Length (bps)] |Distance Traveled During | | | | |Electrophoresis (cm) | |1 |23,130 |4.3642 |1.55 | |2 |9,416 |3.9739 |1.80...