Restriction Mapping of Plasmid DNA

Introduction:

Restriction mapping is the first step to characterizing unfamiliar DNA. Restriction enzymes that digest DNA will reveal how many “cut sites” are available on this DNA. By putting this digested DNA into a gel electrophoresis, we can determine the sizes of the cut DNA fragments.The fragments will run down by a gel electrophoresis, which separates the fragments based upon length of each individual fragment (smaller fragments will travel faster and larger ones are slow). Based on the distance of bands from one another, a plasmid map can be drawn.

Purpose:

There is some shady stuff going on around Palatine. We were approached by the military to characterize some unknown DNA that may or may not have been found at the site of an UFO crash. The purpose of running a gel electrophoresis lab is so that we can gather the needed data to draw a restriction map, and then use this map to characterize the unknown DNA.

Procedures:

Loading the Gel

***Our Gels were made by our teacher, so we did not have to cast them ourselves.

Using a needlepoint pipette, load the contents of each reaction tube into a separate well within the gel. Use a fresh pipette for each reaction tube. Write down the order in which you load the samples.

1. Draw the sample into the pipette
2. Use a steady hand (or two hands) to hold the pipette over the well

3. Make sure there are no air bubbles in the pipette before you load it into the well. An air bubble may cause the DNA to flow out of the well.
4. Position the pipette over the well, dip the pipette very slightly into the well, and slowly expel the sample. Do not punch a hole through the bottom of the well with the pipette.

Electrophoresis

1. Close the top of the electrophoresis chamber, and connect the electrical leads to a power supply. Anode should be attached to anode (red to red) and cathode to cathode (black to black). Make sure both the electrodes are connected to the same channel of the power supply.
2. Turn the power supply and set voltage as directed by the teacher.

3. After the current is applied, the loading dye (bromphenol blue) should move through the gel towards the positive pole of the electrophoresis apparatus.

4. Allow the DNA to electrophorese until the blue bands are about 2 cm from the end of the gel. Your teacher should monitor the process.

***Our teacher monitored the gels, removed them from the chamber, and strained them for us.

Data Analysis

1. Examine your stained gel on a light box or overhead projector.

2. Assign sizes to the lambda DNA/PstI size marker bands on your gel. These marker bands are 514, 805, 1093, 1159, 1700, 1986, 2140, 2450, 2838, 4700, and 11,497 in size. Remember, small DNA fragments migrate more quickly than large ones.
3. Now assign approximate sizes to the DNA fragments of unknown size by comparing them to the lambda DNA/PstI size marker. These approximations will not be perfectly accurate. That is all right since exact sizing is NOT required for determination of the number and relative positions of the cut sites of the restriction enzymes.
4. Determine the total size of the digested DNA by adding up the sizes of the fragments from each digest. You should take an average size from the four digests: pMAP/PstI, pMAP/PstI/HpaI, pMAP/PstI/SspI/, and pMAP/PstI/HpaI/SspI. Remember, the same DNA was digested in each sample, so the fragment sizes should always add up to the same total.

Data:

Analysis:

In order to have sufficient data, we needed to extrapolate some fragments from the lambda DNA/PstI well (we should have been able to see 11, but we only could identify 6). We labeled these lambda DNA/PstI size markers with the given fragment sizes in order to have a gauge to determine the size of the four other wells’ bands. From this, we could estimate the size of the bands, however due to human error (we might've not added all of the DNA into each well, etc.) they were not exactly what we needed to complete a restriction map of the plasmids. All marker lanes (except for the lambda lane, because that's just for sizing purposes) should have a fragment size sum of equal value, since this is the size of the plasmid. In this case, we took the average from all four lanes (3804 kb) and decided to make that our ideal total plasmid size. We then estimated the sizes of the bands (in blue) that would be ideal for the creation of a restriction map, where each lane was equal in total fragment size. All bands and cuts referred to in the following analysis and conclusion are marked in blue above.

In the first well that contains PstI alone, there are two cuts, one making a 3000 kb long fragment, and the other being an 804 kb long fragment. We marked this on a circular plasmid to create a restriction map for PstI by itself, using arbitrary cut locations to demonstrate a hypothetical concept of what the map might look like.

We can see that in the second well, PstI and SspI, together, created 3 cuts, two of which were from PstI (as determined before) and one from SspI. Since the 804 kb fragment from our original PstI well is still present, we can conclude that SspI cuts once within the 3000 kb pair fragment from PstI, creating two smaller fragments (2000 kb and 1000 kb long). On our restriction map, we start with the same two cuts from PstI, but now add a cut within the 3000 kb section in order to show where the cut from SspI might be, separating it into two sections of 1000 kb and 2000 kb.

In the third well, PstI and HpaI, together, created 3 cuts, but this time the 3000 kb long fragment from the initial well PstI was still present. Therefore it can be concluded that HpaI cuts once within the original 804 kb fragment from PstI, creating two shorter fragments of 604 kb and 200 kb. We have demonstrated this in our restriction map by placing an HpaI cut within the original 804 kb strand from PstI, splitting it into two smaller pieces.

In the final well containing all three restriction enzymes, we can see four different fragments. Since we had previously discussed that PstI created two cuts in total and SspI and HpaI had both cut only once when paired with PstI, we can conclude that two of these cuts are due to PstI, one is from SspI, and the other is from HpaI. In the restriction map above, boxed for emphasis, we can see what a plasmid cut with all three restriction enzymes (hypothetically) looks like.

Conclusion:

Since we know that a restriction map of DNA is like a fingerprint for said DNA, knowing the number of cut sites present and their position relative to each other should allow us to identify this mysterious piece of DNA brought to us (which may or may not be, but is most likely, from some sort of extraterrestrial being). Since we obviously work at the best lab ever, we have every resource we could ever need (i.e. Wikipedia). Therefore, we know the recognition sequence and cut site for each restriction enzyme used in this experiment. From this, we can determine the sequence of (some) nucleotide bases in this strand of unknown DNA.

The recognition sequences and cut sites for each restriction enzyme used are as follows.

PstI:

5’ CTGCA|G 3’

3’ G|ACGTC 5’

HpaI:

5’ GTT|AAC 3’

3’ CAA|TTG 5’

SspI:

5’ AAT|ATT 3’

3’ TTA|TAA 5’

By knowing this information and where each cuts on the plasmid relative to each other, we can determine the sequence of nucleotide bases in this particular strand of unknown DNA. Going clockwise from the initial PstI cut on the final plasmid with all three restriction enzymes present (pictured above), the order of the cuts is PstI, SspI, PstI, and HpaI. Therefore, the sequence of DNA must be as follows:

5’ CTGCA|G      AAT|ATT     CTGCA|G      GTT|AAC 3’

3’ G|ACGTC … TTA|TAA … G|ACGTC … CAA|TTG 5’