Lab Summary Of Restriction Enzyme Digestion

Solve the Murder
Koreen Clarke
October 29, 2014
Biology 1 Cellular Processes Lab
Section 903
Tianna Clarke

Materials and Methods
Part I – Restriction Enzyme Digestion
To begin this experiment, the DNA molecules must be cut into smaller fragments with distinct enzymes called Restriction Enzymes through a process called Restriction Enzyme Digestion. Four microtest tubes were labeled 1 through 4 and added 10 µl of Enzyme Reaction Buffer to each of the four reaction tubes using a micropipette. DNA, and Enzyme 1 and 2, were then added to the reaction tubes using a new micropipette tip for each transfer of DNA and enzyme (refer to figure 6.1). The solutions were covered and mixed gently by tapping the reaction tubes on the table which then allowed for the contents to assemble at the bottom of the reaction tubes, followed by incubating the reaction tubes in a 37°C waterbath for 45 minutes. Finally, after the incubation was complete, 5 µl of 10x gel loading solution was added to each reaction tube to stop the reaction. Once again, the reaction tubes were covered and tapped gently to mix the solution and stored in the refrigerator for gel electrophoresis.
Table 6.1 Summary of Restriction Enzyme Digestion
Reaction Tube Reaction Buffer DNA 1
(µl) DNA 2
(µl)
Enzyme 1
(µl)
Enzyme 2
(µl)
Final Volume
(µl)

Crime
Scene
Samples

X
--- 45*

Suspect 1

Suspect 2

Part II – Casting and Loading a Gel for Electrophoresis
The second part of this experiment consists of Agarose Gel Electrophoresis, a method used to separate an assorted population of DNA or proteins in a solution of agarose. The DNA can be separated by charge and/or length of the DNA fragment. The casting of the gel was prepared by adding .5g/50mol of agarose and 50 ml of 1 x TBE buffer into a 250 ml flask and then swirled to diffuse the clusters of agarose powder. The flask was then covered with plastic wrap to reduce evaporation, and heated in a microwave temporarily for one minute making sure the flask did not boil over. The solution should be observed to be a clear substance having no undissolved particles and set aside to cool. After placing the gel comb at one end of the tray, and then placing the tray securely into the gel box, the solution was poured into the tray where it would become firm and cool to touch roughly around 20 minutes. After the gel is completely solidified, 250ml of distilled water was poured to completely submerge the gel, and then 10 µl of Bromophenol Blue was loaded into the wells of the gel.
Part III – Analysis of Crime Scene Samples In this part of the experiment, the DNA will need to be stained with ethidium bromide. This is necessary because DNA is invisible and cannot be seen on a gel without staining. Ethidium Bromide intercalates itself with thymine base pairs which illuminates under ultra violet light. Wearing gloves, as ethidium bromide has carcinogenic characteristics, is added to a solution consisting of .4g/50ml of agarose and 50 ml of buffer after being heated until the agarose was completely dissolved, and then added to the gel tray in the gel box, placing the comb at one end as previously done in Part II of this experiment. After solidified, the gel was removed along with the tray from the gel box and relocated so that the row of wells will be at the negative (black) pole. The electrophoresis device was afterward filled with 250 ml of 1X TBE buffer and the gel comb was removed. 30 µl of each of the DNA samples was loaded to each correctly marked tube in the following manner:
Lane Tube
1 Markers Standard DNA Fragments
2 CS 1 DNA from crime scene cut with Enzyme 1
3 CS 2 DNA from crime scene cut with Enzyme 2
4 1 DNA from Suspect 1 cut with Enzyme 1
5 2 DNA from Suspect 1 cut with Enzyme 2
6 3 DNA from Suspect 2 cut with Enzyme 1
7 4 DNA from Suspect 2 cut with Enzyme 2
8 Markers Standard DNA Fragments

After the DNA samples were loaded, the apparatus was covered, the black/red wires were plugged into the correctly color coded power sources and turned on to run at 150V and 150mA for 40 minutes; the gel was removed and placed on the UV illuminator after being covered with plastic wrap. The bands on the gel were examined, analyzed and compared to match with the crime scene samples.

Results

As the electrophoresis reactions are occurring, the negative current is going to push the bands toward the positive end. Each line in figure 6.2 represents a band of DNA. The fragments that are smaller are going to travel a longer distance than the fragments that are larger. The way that agarose is designed and how concentrated it is affects the molecules that are trying to pass through the solution. If the agarose concentration is less concentrated, the larger fragments will have no problem moving through, where as if the solution was more concentrated, the larger fragments will have more of a problem than the smaller fragments causing them to travel a shorter distance. The length of an unknown DNA fragment may be projected by comparison to an average sample of DNA, also known as a marker, encompassing a variety of differently sized DNA fragments. The bands are more similar in length moving across the row. In Figure 6.2, the two markers are lane 1 and lane 8. Lane 2 represents the crime scene DNA with Enzyme 1. Lane 3 represents the crime scene DNA with Enzyme 2. Lane 4 and 5 represent the DNA of Suspect 1 with Enzyme 1 and 2. Lane 5 and 6 represent the DNA of suspect 2 with Enzyme 1 and 2.
Figure 6.2 shows that the first and second bands in lane 2, lane 4, and lane 6 all match. There are also matches between lanes 3 and 7, as bands one and two are identical to one another. The DNA that was found from crime scene cut with Enzyme 1 was identical to both Suspect 1 and Suspect 2 DNA cut with Enzyme 1. The DNA from crime scene cut with Enzyme 2 was also matched with the DNA from Suspect 2 cut with Enzyme 2.
Discussion
During the analysis of the data, it became evident that suspect 2 is indeed the criminal. When analyzing the results, you can see from figure 6.2, that lane 2, which is the DNA from the crime scene cut with enzyme 1, is identical to lane 4, which is the DNA from suspect 1 cut with Enzyme 1, and also lane 6, which is the DNA from Suspect 2 with Enzyme 1. The precision of the bands at the same fragment length and spot on the electrophoresis gel lets me know that suspect 1 and suspect 2 are twins. Due to the fact that there was also matches in lane 3, which is the DNA from crime scene cut with Enzyme 2, and lane 7, which is the DNA from suspect 2 cut with Enzyme 2, Suspect 2 is guilty. Two enzymes are used throughout this process to make sure the results are undisputable. If only Restriction Enzyme 1 had been used, then both suspect1 and suspect 2 would have been guilty for the crime as both their DNA bands were identical. Given that Suspect 2’s DNA was found at the crime scene does not necessarily mean that the suspect is guilty, however that they were definitely around the crime scene before/after the event of the crime.
Possible experimental problems such as not adding the proper amount of DNA and enzyme to the reaction tubes during the Restriction enzyme digestion, not staining the DNA and/or not allowing the DNA to incubate for the suitable amount of time could have invalidated the results.
Gel electrophoresis is used in day-to-day activity in various ways such as maternity/paternity testing, and genotyping, testing for heritable diseases. DNA typing is also used in Identifying human remains. Scientists have used DNA profiling to verify or refute that the person in the grave was or was not, the person who was believed to be there. DNA typing can likewise be manipulated to identify food, for example, fish supplied in sushi meat was in actuality from dolphins and
whales.