DNA Profiling Using Agarose Gel

DNA Fingerprinting using Gel Electrophoresis

 In the experiment described below two suspects DNA is being compared to the DNA found at the scene of a crime. The DNA suspect samples are being compared and analyzed using the process of gel electrophoresis and the science of DNA fingerprint to determine which DNA best matches the culprit. By introducing the suspect and crime scene DNA to 2 different restriction enzymes they will be cut into readable segments that can then be compared and contrasted to find the best match. This DNA/enzyme solution is injected into the pores of an Agarose x EtBr gel and introduced to an electric current to separate out different sized fragments to produce DNA ladders that will then be compared to a standard DNA sample as well as the crime scene DNA (Upadhyaya, Exercise 6/7). This data is then used to help scientist determine which suspects DNA is or is not found at the crime scene. This practice is done through the principles of DNA fingerprinting which maps out an individuals genetic makeup to determine different aspects of their genome or in this case whether it in fact matches that of an unknown sample (Your Genome, 2).

Introduction:

 DNA is useful to scientists in many different ways and for many different applications. Genes for example are “the basic physical and functional unit of heredity” (U.S. National Library of Medicine, 1). Each person receives two copies of each gene in our genetic makeup, one from their mother and one from their father, and these sequences of DNA help to determine different traits in our physical and genetic characteristics.  More specifically, some genes contain different variations of themselves called alleles. This can be attributes to small mutations or changes in the genetic sequences causing the different variety of outcomes (U.S. National Library of Medicine, 2). For example, eye color is a gene that has many different alleles such as blue, green, brown, and hazel. Theses sets of characteristics are unique to each individual making no two people alike.  People within the same family, siblings or even twins for example, have a similar genetic makeup but have many different combinations of the same sequences making even their DNA different.  The process of genetic changes can often be related to the idea of genetic mutation. This occurs when small permanent changes in the genetic code occur, and are possible in all gene sequences. Genetic mutation fall into two major categories; hereditary and acquired. Hereditary mutations are inherited from parent to child and are experienced throughout the child’s entire life. On the other hand, acquired mutations occur during a persons lifetime and are not changes that they will pass along to their children. These mutations effect a group of specific cells and can often be traced back to environmental factors or mistakes made during the process of DNA replication (What is a gene mutation, U.S. National Library of Medicine).

 DNA fingerprinting or DNA profiling is the process used to identify an individual based off of a sample of DNA and by analyzing genetic makeup and can be used for many different purposes in genetics today (What is a DNA fingerprint, 1). These pieces of DNA are broken down using restriction enzymes, they identify certain sequences and chains within the makeup of DNA and cut it into these smaller sequences to be read and interpreted. When cut, these enzymes produce two different ends on the DNA; a jagged and a blunt end. These jagged or “sticky” ends can later be used for processes such as cloning and are linked back together using a different enzyme known as DNA ligase. On the other hand, blunt ended segments can be reattached just not as easily as ones with sticky ends, but they similarly use DNA ligase for this process. Each restriction enzyme has a unique set of 4-5 base pairs that will indicate and initiate the process of cutting for its specific purpose. More specifically, in this experiment the Restriction Enzymes EcoRI and HindIII were used to break down two different suspects DNA, both of theses restriction enzymes create “sticky” ends thought they are cutting the DNA into different segments based off of the set of base pairs that indicate their use (Upadhyaya, Exercise 6). 

 DNA fingerprinting and restriction enzymes are all ideas that help make the process of gel electrophoresis possible. Gel Electrophoresis is the separation of biological molecules in substances such as DNA, RNA, and proteins, using an electric current so they can be read and analyzed. Due to the charge and size of these molecules, we are able to use pores in an substances like Agarose gel to separate them into a way that can be used to help match like segments as well as determine different characteristics about these molecules (Upadhyaya, Exercise 7). Being that DNA is the biological molecule being separated in this experiment, its negative charge and different sized segments due to the cutting done by the restriction enzyme discussed above, we are able to compare the segments found in the two suspects in question and compare them to the segments found in the DNA at the crime scenes.

Materials and Methods:

DNA Dissection: With a group of 2-4 students, 4 aliquot contains were obtained and labeled 1 through 4. A micropipette deposited 10µl of the Enzyme Reaction Buffer to each of the four test tubes. It is crucial to ensure that all of the liquid is settled at the bottom of the tube. DNA and Restriction Enzyme were then inserted into tubes 1-4 with the micropipette based off of the table below. A fresh tip was placed onto the pipette for each substance to prevent contamination.

Tubes 1-4 were then capped after the introduction of the DNA and enzyme and then tapped to mix. The tubes were incubated for 45 minutes at 37*C. Once incubation was complete, the samples were stored until electrophoresis took place (Upadhyaya, Exercise 6). 

Pouring and Loading Gel Electrophoresis: The agarose concentration that was utilized for this experiment was an 0.8%. (0.4g/ 50mL) The agarose was measured using an electric scale and poured into a 250mL flask with 50mL of 1xTBE buffer. First the mixture was swirled to break down any large clumps and then the flask was covered with a 50mL beaker to avoid any evaporation. The mixture was microwaved for approximately 1 minute and then left to cool. While waiting for the agarose solution to cool, the electrophoresis tray was set up. The comb was placed at one end of the gel box and then wedged into the electrophoresis tray, an airtight seal was created. Once the flask was cool to the touch, the TA added 2.5µl of Ethidium Bromide (EtBr) and the solution was then poured into the tray and left to set. (Ensure that the tray is not bumped or jostled as this can affect the setting of the gel.) After approximately 20 minutes the gel will be cool an become firm to the touch. If the gel is not completely firm, then more time should be allowed for it to cool before continuing. The gel box was then removed from the tray and turned so that the open ends are facing the reservoirs on either side of the tray and the comb at the end of the gel was removed. (The pores must be positioned at the same side as the negative pole of the current apparatus.) The electrophoresis tray was then filled with approximately 250mL of 1xTBE buffer or just enough to coat the surface of the agarose x EtBr gel. 5µL of 10x gel were then added to tubes 1-4, capped, and mixed by tapping. 15µL of each solution were added into the pores in lanes 1 through 8. The key is located below (Upadhyaya, Exercise 6/7).

Running Electrophoresis: Once loaded, the slide cover was clicked into place on the gel box. The plugs were then connected to the power box (Black to black, red to red) and the box was plugged into a power source. (Double check that the pores are located on the black or negative side of the current). The current was run at 150V and 150mA for approximately 30-40 minutes. The current can be detected by the presence of bubbles on the sides of the electrodes. After the allotted time frame, the power source is unplugged and the cover was removed from the gel box. With gloved hands, the gel was removed from the electrophoresis mechanism and placed onto plastic wrap on the cover of a UV illuminator box. Using the UV light, the bands were then examined and compared to the DNA from the crime scene tubes. Lanes 4 and 6 were compared to lane 2 as they were all cut using restriction enzyme 1 and lanes 5 and 7 were compared to lane 3 as these samples were all cut using restriction enzyme 2. A photo of the gel was taken and analyzed for results. This picture is listed below under the results section. The agarose x EtBr gel was then discarded into the hazardous waste (Upadhyaya, Exercise 7). 

Results and Data:

 Once the electric current was applied to the Agarose gel, the negatively charged DNA migrated towards the positive end of the current and separated based off of the size of the base pairs and segments of DNA that were created by the interaction between the Restriction enzyme and the suspect and crime scene DNA. These bands of DNA create a ladder and each rung represents a different sized fragments, the larger being closer to the pores and the smaller migrating further away. This ladder is then interpreted using figure 7.1 from the lab manual and the marker or standard DNA that can be found in lanes 1 and 8 which can be used as a reference (Upadhyaya, Exercise 7).

 When checking the electrophoresis results, the restriction enzyme used is crucial to help interpret the data shown in the image above. The pore CS1, is the DNA that was found at the crime scene that has been introduced to Restriction Enzyme #1. This means that the DNA from suspects 1 and 2 that are located in the pores 4 and 6 were also brought into contact with this restriction enzyme 1 and therefore have been cut at the same recognition site. Based off of Figure  7.1 in our lab manual, the DNA present in pore 2 has bands that represent 1000 and 1650 base pairs. When compared to the suspect data, suspect 2’s DNA correctly matches the same number of base pairs while suspect 1 shows a band around 1650 base pairs and a fair trace around 1000 but does not match the DNA at the crime scene.

 Likewise, the DNA present in pore 3, was DNA found at the crime scene after it has been cut using Restriction Enzyme 2. Referring back to Figure 7.1 in the lab manual, this lane shows about 2000 base pairs and will be used to interpret the DNA that is taken from suspect 1 and 2 that have also been introduced to Restriction Enzyme 2 and can be found in lanes 5 and 7. The DNA taken from suspect 1 shows a mark on the ladder that represents 1650 base pairs, and DNA from suspect 2 similar to the crime scene DNA shows a ladder mark of 2000 base pairs. Unlike restriction enzyme 1, enzyme 2 only cut the DNA to create one mark on the ladder.

Figure 1:

The image above is an example of the gel electrophoresis process described in the methods and material section and can be used to describe the results listed above.

Discussion:

 Based off of the results discussed above it can be determined that suspect 2’s DNA matched the crime scene DNA cut with both Restriction Enzyme 1 and 2. This does not necessarily mean that suspect 2 is guilty. Between the possibility of error as well as limitation of restriction enzymes used, the results don’t determine the suspect as guilty but help instead narrow down the suspect DNA that fit the genetic profile that was found at the scene of the crime. These results can then be used in support of other evidence to help convict the suspect.

 There a few different examples of errors that could have effected results or changed the overall outcome of the gel electrophoresis. In the preparation of the tubes 1-4, there was a potential contamination of tube 3 with the DNA from suspect 1. There was no solution deposited but the tip that had been used to deposit DNA 1 into tubes 1 and 2 was not removed before entering the mouth of tube 3. It was then realize and then removed and changed before DNA 2 was deposited into tube 3. The transfers of solution were made using a micropipette as covered in the materials and methods section, though this creates an accurate measurement there are often cases where not all of the solution becomes deposited and therefore the balance of DNA to enzyme could have been slightly off causing a skew in results. This micropipette could have also caused some of the solution to be spilled into the buffer solution that was used to cover the Agarose x EtBr gel causing a smaller amount of DNA to migrate through the gel. Lastly, when applying the plate that contained the positive and negative electrodes to the electrophoresis tray, the first lid was not working when it was pulled into the power source causing the gel box to have to be removed and reinserted into the tray in order to have the pores on the same side as the negative electrode of the new lid that was used, this could have caused a shift of the amount of concentration located within the pores or even displaced some of the buffer solution. All of these errors thought minute, could account for small changes in the overall results of the electrophoresis gel.

 While DNA fingerprinting is a practice that is most widely known to be used in the process of solving crimes, as seen in the experiment above, it has also taken on a new role more recently in the Obstetrics and Gynecology department of medicine. Pregnancy is an area of medicine that over the past few decades has experienced a new set of practices that help not only the doctors but also parents learn more about the baby prior to birth. One type of testing that can be done is known as cell-free DNA testing, the placental DNA contains the same DNA as the unborn fetus, and these cells can be taken from samples of the mothers blood and analyzed using DNA fingerprinting to screen for genetic abnormalities, as well as diseases that can be traced using genetic markers. Similar to the experiment discussed in this report, the placental DNA is then compared to a standard DNA to determine wether they contain similarities to the indicators for the genetic factors being tested (American College of Obstetrics and Gynecology, 7). I have personally become very familiar with this process as I am currently 18 weeks pregnant and have had this genetic testing done for my baby. It is fortunate that these kinds of technologies have become available because a lot of this process was forced to wait until after the baby was born  or through much more invasive measures for both mother and baby, and now parents and physicians can be informed much sooner during the gestation process.

 In order to compare the segments of DNA used for DNA fingerprinting in examples such as this lab as well as in real-world application such as the pre-natal genetic testing discussed above, different restriction enzymes are utilized to cut the DNA into different fragments as each enzyme has its own recognition site and therefore severs the DNA in different places creating different sized fragments. Since two restriction enzymes were used we are able to compare the suspects DNA to two different sets of fragments from the DNA that was found at the crime scene. If only one of the enzymes had been used we would only be able to compare one set of fragments between the suspects and crime scene DNA which would not provide as conclusive results as the use of both restriction enzymes. Overall the design of this experiment was used to create the best data to help prove which suspect would best fit the DNA found at the crime scene.

References:

• DNA Fingerprinting – GeneEd – Genetics, Education, Discovery. U.S. National Library of Medicine. https://geneed.nlm.nih.gov/topic_subtopic.php?tid=37&sid=38 [accessed 2018 Oct 26].
• Upadhyaya, A. 2018. Exercise 3: Diffusion and Osmosis. In: Cellular Processes (Conner, S., Cleveland, A., Buzzeo, R. etal) USF, Tampa, 33.
• What is a DNA fingerprint? Facts. 2016 Jun 2 http://www.yourgenome.org/facts/what-is-a-dna-fingerprint [accessed 2018 Nov 2].
• What is a gene? – Genetics Home Reference – NIH. U.S. National Library of Medicine. https://ghr.nlm.nih.gov/primer/basics/gene [accessed 2018 Oct 26].
• What is a gene mutation and how do mutations occur? – Genetics Home Reference – NIH. U.S. National Library of Medicine. https://ghr.nlm.nih.gov/primer/mutationsanddisorders/genemutation [accessed 2018 Oct 28].
• Women’s Health Care Physicians. Prenatal Genetic Screening Tests – ACOG. https://www.acog.org/Patients/FAQs/Prenatal-Genetic-Screening-Tests [accessed 2018 Nov 1].