Restriction Enzymes and Electrophoresis

Introduction
When talking about restriction enzymes, it is necessary to first talk about viruses. Viruses are small packets of protein and DNA that are not actually classified as lifeforms for a few reasons. Most prominent of which, viruses cannot reproduce. In order to keep the species alive and pass on genetic material to offspring, viruses infect other cells, either prokaryotic or eukaryotic, and insert their DNA into the DNA of that cell. Then the cell is forced to produce large amounts of the viral proteins and create many copies of the original virus inside of the cell until its usefulness runs out and the cell is killed by the viral DNA, allowing the new viruses produced to spread and infect others.

As a defense mechanism against viral attacks, many bacteria and eukaryotes have developed what are called restriction enzymes, or proteins that cut DNA. Therefore, when a virus inserts its DNA into a new host, that host's restriction enzymes would cut the DNA into fragments, not allowing the cell to become infected. The restriction enzymes only work at sites on the viral DNA called palindromes, or places where the base pairs are the same in one direction as they are in the opposite direction on the corresponding strand.

Restriction enzymes have become an important part of gene splicing by making it possible for scientists to connect two sets of genes of different organisms or replace harmful genes that a person has with healthy genes. Also, through a process called gel electrophoresis, in which DNA fragments are run through gel by a current, similarities or heredity can be found between people, being an important tool for paternity testing among other things.

During the week of February 8th, the lab group of Shreyan,  Mark, Vinay and Vikram returned to the lab to perform DNA cutting using restriction enzymes and also gel electrophoresis. Through this process, the group should be able to determine the size of the DNA fragments cut by the enzymes. The group was tasked with using lambda virus DNA, which is about 50,000 base pairs long.

Procedure
On the first day of the week, we gathered our materials together and then set to work. We had 3 microtubes of different restriction enzymes which were called; PstI, EcoRI, and HindIII. We also had a microtube full of uncut lambda DNA strands. For our four experiment microtubes, one we filled with Lambda DNA and a restriction buffer, and the other were filled with Lambda DNA, restriction buffer, and one of the enzymes listed above. The tubes were labeled P, E, H, and L, and below is a table of their contents.

Tube	Lambda DNA	Restriction Buffer	PstI	EcoRI	HindIII
P	4 µl	5 µl	1 µl	0 µl	0 µl
E	4 µl	5 µl	0 µl	1 µl	0 µl
H	4 µl	5 µl	0 µl	0 µl	1 µl
L	4 µl	6 µl	0 µl	0 µl	0 µl

After this we spun the tubes in a centrifuge to mix the contents completely. Having mixed the contents of each tube, the group left the tubes overnight so that the restriction enzymes could do their work.

On the second day, I was unfortunately absent due to illness, but Vinay, Shreyan and Vikram continued the experiment without me. The second day was the gel electrophroesis day. The group obtained marker DNA from our teacher, and the function of the marker DNA is to be completely split up by the enzymes we were using in order for us to estimate the length of the strands of DNA used in our experiment. The marker DNA, as well as the other DNA and restriction enzyme samples that we had, were loaded into agarose gel, pictured below. The agarose gel that was used was actually clear, but the picture below is of the gel after it has been stained so that we could see the distance traveled by the DNA fragments.

When we are ready to begin the electrophoresis, a current will be run through the gel, and because it is a negatively charged acid, the DNA will flow towards the positive electrode. However, the gel acts as a viscous buffer to the DNA, so not all of the DNA will flow to the positive electrode quickly. Instead, smaller fragments of DNA pass through the gel easier and will travel further towards the positive electrode than larger fragments of DNA.

The agarose gel, with the DNA fragments loaded up inside of it, was then put into the electrode apparatus, pictured below, and a current was run through the gel for about 20 minutes.

After the gel was removed from the electrophoresis apparatus, the gel was dunked in fast acting blue dye overnight so that the DNA bands were visible. The process by which the gel was stained is shown below.

On the third day, results were gathered and the lab group measured the lengths that the DNA traveled down the gel from the DNA wells that all of the fragments started in. This is shown below.

Data/Observations
Unfortunately, because I was absent for all but the first day of the lab, I was not able to gather data with my lab group. But, after explaining what the data meant, and also a discussion with my teacher Mr. Wong, I was able to understand the data set that resulted from the experiment.

Using the data gathered in this experiment, the group was tasked with estimating the length of the DNA strands cut by each individual restriction enzyme. To do this we had to compare the bands that we saw in our agarose gel to lengths traveled by fragments that were in the marker group. These strands were known to have a certain base pair lengths, so we could estimate the lengths of our DNA fragments through comparison and a little bit of educated guesswork. Unfortunately, our groups's marker DNA lane was not recorded correctly or simply did not perform as it was meant to, so the DNA lengths were borrowed from our classmates.

Here is a table of the distances traveled by each lane, including the marker DNA lengths obtained from our peers:

	M (Marker DNA)		L (Uncut DNA)		P (PstI)		E (EcoRI)		H (HindIII)
Bands	Distance in mm	Actual base pairs	Distance in mm	Estimated Base Pairs	Distance in mm	Estimated Base Pairs	Distance in mm	Estimated Base Pairs	Distance in mm	Estimated Base Pairs
1	14	23,130	10.2	50,000	15.2	20,000	15.2	23,000	15.2	21,000
2	16	9,416			19.1	6,000	19.1	6,000	17.8	7,000
3	18	6,557			20.3	4,000
4	22	4,361
5	27	2,332
6	Not visible	2,027

Using simple guesswork and estimation, the group assigned values of base pair lengths to the DNA fragments that were cut using restriction enzymes. But is there a better way to estimate fragment lengths? A second way that the group estimated the lengths was using what is called a semilog graph. Using the info from the marker DNA group, we created a graph relating distance traveled to base pair length of the DNA strands by plotting the points of the marker strand and creating a line of best fit. This graph is shown below. Then, by using the graph, the group was able to estimate lengths of the DNA fragments somewhat easier.

Using this graph, we estimated the base pair length of each DNA fragment, shown below.

	M (Marker DNA)		L (Uncut DNA)		P (PstI)		E (EcoRI)		H (HindIII)
Bands	Distance in mm	Actual base pairs	Distance in mm	Estimated Base Pairs	Distance in mm	Estimated Base Pairs	Distance in mm	Estimated Base Pairs	Distance in mm	Estimated Base Pairs
1	15.2	23,130	10.2	55,000	15.2	18,000	15.2	18,000	15.2	18,000
2	17.8	9,416			19.1	8,000	19.1	8,000	17.8	10,000
3	19	6,557			20.3	6,000
4	22	4,361
5	27	2,332
6	Not visible	2,027

As you can see, some of the values are extremely different in the new table of estimations.

Conclusion
I believe that the experiment can be considered a success. Though our marker DNA did not turn out as planned, we were still able to create our semilog graph and also get some good estimations of the length of our other DNA fragments. I am not completely sure whether or not any of our results are correct and that is the actual length of each fragment, but I am willing to bet we got somewhat close to the right answer. I know that our estimations for the uncut lambda DNA, about 50,000 base pairs is correct, and I think the cuts made by PstI and EcoRI were valid too. I am not sure whether or not the cuts made by HindIII, or at least the data we recorded from it, were valid. I think I remember reading somewhere that the lambda DNA is cut in many places by HindIII, but it was only cut into two fragments in our experiment.

I believe the only error made during this experiment was in collecting data from the agarose gel. I know that the data we gathered regarding the marker DNA was wildly incorrect, so much so that had to use another group's data to get the correct graph. I am also skeptical about the results gained from the HindIII cuts, as mentioned above. I think that when looking at the agarose gel, it is necessary to shine light from the bottom up through the gell so the dyed lines of DNA are extremely clear and can be recorded easily. I also think that when graphing the semilog graph, it would have been easier to graph on paper rather than the iPad so that we may use a rule and get better, more accurate results.