I. Title. Restriction Enzyme Mapping of pBR322 Using Agarose Gel Electrophoresis. II. Authors. Author: Partner: Section: Thursday, 1:10 pm Date of Experiment: October 25, 2012 III. Introduction. 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.
IV. Results. 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 | |3 |6,557 |3. 8167 |1. 95 | |4 |4,361 |3. 6396 |2. 20 | |5 |2,322 |3. 3659 |2. 95 | |6 |2,027 |3. 3069 |3. 15 | |7 |564 |2. 7513 |6. 00 | 3. Graph I (Standard Curve). As discussed in the introduction above, the distance traveled by a DNA fragment toward the anode in agarose gel electrophoresis has a linear relationship with the log of its length (in base pairs).
The graph attached as Exhibit B is a scatter plot of the data from the final two columns of Table 1 above, with a trendline showing the relationship between the variables. Note that data for fragment greater than 5,000 base pairs in length are shown on the plot but were not used in calculation of the trendline as a 1% agarose gel was used during electrophoresis and such a gel concentration does not effectively resolve fragments of lengths greater than 5,000 base pairs. 4. Table II. The following table presents the data obtained by performing electrophoresis on the DNA fragments from the pBR322 restriction enzyme digests.
|Band # |Distance Traveled During Electrophoresis (cm) |Log[Fragment Length (bps)] |Fragment Length |Normalized Fragment | | | | |(bps)] |Length (bps) | |EcoR1 (E) | |1 |2. 00 |3. 1 |4107 |4480 | |Total bps |4107 |4480 | |HincII (H) | |1 |2. 55 |3. 49 |3105 |3199 | |2 |4. 35 |3. 09 | 1243 |1281 | |Total bps |4348 |4480 | |PvuII (P) | |1 |2. 00 |3. 61 |4107 |4480 | |Total bps |4107 |4480 | |EH | |1 |2. 50 |3. 50 |3185 |3288 | |2 |5. 50 |2. 84 |693 |716 | |3 |6. 30 |2. 66 |461 |476 | |Total bps |4339 |4480 | |EP | |1 |2. 95 |3. 40 |2534 |2354 | |2 |3. 15 |3. 36 |2289 |2126 | |Total bps |4823 |4480 | |HP | |1 |3. 35 |3. 32 |2067 |1897 | |2 |3. 85 |3. 21 |1603 |1471 | |3 |4. 40 |3. 08 |1212 |1112 | |Total bps |4882 |4480 | |EHP | |1 |3. 35 |3. 32 |2067 |1940 | |2 |3. 85 |3. 21 |1603 |1504 | |3 |5. 55 |2. 83 |676 |634 | |4 |6. 45 |2. 3 |428 |402 | |Total bps |4774 |4480 | 5. Restriction Enzyme Map. Attached as Exhibit C is a restriction enzyme map of pBR322 showing the relative locations of the restriction sites and the distances between them.
IV. Discussion. The first part of the analysis consisted of plotting the log of the distance traveled during electrophoresis by the lambda DNA fragments resulting from the cleavage of lambda DNA by HindIII (data summarized in the last two columns of Table I). Such plot is shown on Exhibit B, with an overlay of the linear relationship between the log of the number of base pairs in a fragment and the distance such fragment travels during electrophoresis.
Based on the foregoing relationship, the number of base pairs in each fragment resulting from the restriction enzyme digests of pBR322 was calculated (as shown in the fourth column of Table II). The number of base pairs in all fragments resulting from each digest were then added together, providing seven estimates of the total number of base pairs in pBR322, ranging from 4,107 to 4,882 (each sum shown in Table II next to “Total bps”). Since there was no reason to believe that any one digest would provide a more accurate estimate of the total number of base pairs in pBR322 than any other digest, an average of the seven estimates was used (i. e. , 4,480 base pairs).
Then, in order to more easily compare fragment lengths and deduce the relative locations of the restriction sites, each calculated fragment length was normalized to a plasmid length of 4,480 base pairs and such normalized fragment lengths are shown in the final column of Table II. The photograph attached as Exhibit A-2 shows the results of the electrophoresis, with each lane marked with a letter or letters representing the restriction enzyme(s) digest used in such lane (with “(” representing the lambda DNA/HindIII digest and “pBR322” representing a sample of the uncut plasmid) and each band representing a DNA fragment. The “E” and “P” lanes each show a single fragment, indicating that there is only one restriction site for each of EcoR1 and PvuII on the plasmid, as a circular plasmid cut at one site will yield a single fragment.
The “H” lane, on the other hand, shows two fragments, indicating two restriction sites for HincII on the plasmid. The two fragments in the “H” lane are relatively far apart, indicating that one of the fragments is significantly larger than the other and the lengths of such fragments are shown on Table II. The “EH” lane shows three fragments, indicating three restriction sites (one for EcoR1 and two for HincII). The first fragment traveled about the same distance as the first fragment in the “H” lane, while the second and third fragments each traveled farther than the second fragment in the “H” lane (meaning that they are each smaller than the second “H” fragment).
Further, as shown on Table II, the calculated base pair length of the second “H” fragment is reasonably close to the sum of the base pair lengths of the second and third “EH” fragments, indicating that EcoR1 cuts the smaller of the two fragments created by HincII into two unequal fragments, one being about 20% larger than the other. The “EP” lane shows two fragments, indicating two restriction sites (one for EcoR1 and one for PvuII). While the fragments are clearly distinguishable, they are relatively near to one another, indicating fragments nearly equal in size, and Table II shows that one fragment is approximately 10% larger than the other. Thus, EcoR1 and PvuII have restriction sites on opposite sides of the plasmid (though not diametrically opposed). The “HP” lane shows three fragments, indicating three restriction sites (two for HincII and one for PvuII).
The third fragment traveled about the same distance as the third fragment in the “H” lane, while the first and second fragments each traveled farther than the first fragment in the “H” lane (meaning that they are each smaller than the first “H” fragment). Further, as shown on Table II, the calculated base pair length of the second “H” fragment is reasonably close to the sum of the base pair lengths of the first and second “HP” fragments, indicating that PvuII cuts the larger of the two fragments created by HincII into two unequal fragments, one being about 30% larger than the other. To complete the pBR322 digest lanes, the “EHP” lane shows four fragments, indicating four restriction sites (one for EcoR1, two for HincII and one for PvuII).
Comparing the “EHP” bands to the “HP” bands shows that the first two fragments are of approximately the same length (having traveled approximately the same distance), while the third “HP” fragment (which is the same as the second and smaller “H” fragment) appears to have been cut into two smaller fragments of unequal size. Such cut must have been made by EcoR1, confirming that the enzyme cuts the smaller of the two fragments created by HincII. Conversely, comparing the “EHP” bands to the “EP” bands shows that the last two fragments are of approximately the same length, while the first “EH” fragment (which is the same as the first and larger “H” fragment) appears to have been cut into two smaller fragments of unequal size.
Such cut must have been made by PvuII, confirming that the enzyme cuts the larger of the two fragments created by HincII Synthesizing the foregoing information yields the restriction enzyme map of pBR322 that is attached as Exhibit C. The numbers of base pairs between the restriction sites are based on the normalized lengths of the fragments from the “EHP” digest, as shown in Table II (rather than on averages for fragments across digests, which would have proved difficult to reconcile with the estimated total length of the plasmid). Despite containing only the uncut plasmid, the “pBR322” lane shows two bands that traveled different distances towards the anode during electrophoresis.
Such bands can be explained by the plasmid taking on two different conformations, perhaps supercoiled and open-circular, that travel at different rates through the agarose gel “sieve”. This experiment could have been expanded by introducing additional restriction enzymes, creating a more detailed map of the pBR322 plasmid. Restriction enzymes maps of the type produced in this report have a variety of applications. For example, they can be used in comparing the relationship between the genomes of different organisms, in DNA sequencing, and as references in engineering plasmids. Restriction enzymes also play an important role in the construction of recombinant DNA in gene cloning experiments.