Dna Fingerprinting - Lab Experiment

Caught by My Genes Abstract DNA fingerprinting is one of the most advanced forms of technology we have today. With this advancement, comes great benefits and discoveries that can help society. The objective of this experiment was to discover the guilty suspect of a crime using gel electrophoresis and restriction enzymes. The use of agarose gel coupled with electrophoresis made it possible to visibly observe separation bands between the suspect’s DNA, cut with two restriction enzymes, and the DNA from the crime scene, also cut with two restriction enzymes. After comparing the bands, it was concluded that suspect 2 was the guilty individual because their DNA (cut with both enzymes) matched both crime scene samples. Introduction DNA, or deoxyribonucleic acid, is a double helix molecule found in all cells (Upadhyaya, 2017). DNA is comprised of two polymer strands that coil around to form the helix shape, while each polymer strand, also known as a polynucleotide, is composed of a nitrogenous base, a deoxyribose sugar and a phosphate group. The sugar and phosphate group work together to form the skeleton of the strands and provides a structure for the base pairs to connect. In every base pair, there is one single-ringed pyrimidine base, which are cytosine and thymine, and one purine base, which are adenine and guanine (Upadhyaya, 2017). However, cytosine (C) only pairs with guanine (G), and adenine (A) only pairs with thymine (T). Both base pairs attach with the nitrogenous base using hydrogen bonds, 3 and 2, respectively. Furthermore, when a cell reproduces through cell division, the DNA is duplicated based on its complementary base-pairing (Upadhyaya, 2017). So, for example, if the sequence of one DNA strand is 5’AGCTAGC-3’, its complementary strand would be 3’-TCGATCG-5’. Zooming out of the inside structure of DNA, its function is equally important. The sequence in which the base pairs are situated can determine what genetic information is ‘built’ for the organism. A particular section of DNA that determines a specific characteristic in an organism is known as a gene (Urry et al, 2017). There are also alternate forms of genes that are different in the base sequences that can result in changes of the genetic traits known as alleles (Urry et al, 2017). While everyone has DNA and genetic information, no two organisms can have the same DNA. This is primarily due because genes are inherited from the parents, one copy from the mother and one copy from the father, this gives the individual their own unique genetic makeup or DNA fingerprint. In addition to individuals having their own DNA fingerprint, it may be hard to find any variation in the sequences, which is why restriction enzymes are used. With the help of these enzymes, differences in the DNA can be visualized. They work by ‘scanning’ the DNA strand looking for a specific location, or recognition site, which will typically consist of about 4-8 nucleotides. After finding the wanted section it will then cut the strand, also known as enzyme digestion, in to half or jagged leaving hangs or ‘sticky ends’ (Vincze et al, 2003). In this experiment, the method of gel electrophoresis and restriction enzymes were both used to best separate specific DNA sequences for comparison. Electrophoresis is a technique that uses an electric field to separate charged particles in an agarose gel (Upadhyaya, 2017). Since DNA contains phosphate groups, which has a negative charge, in its skeleton, during electrophoresis the stained DNA would slowly migrate towards the positive pole leaving behind DNA fragments. Once the electrophoresis is completed, the resulting fragments left behind by the DNA can be further examined and compared to the DNA fragments from the crime scene, which can be used to discover which suspect did the crime. Likewise, the two restriction enzymes, EcoRI and HindIII, were used to cut the DNA samples from both suspects in separate reactions, they were then compared to the samples at the crime scene after the electrophoresis. Materials and Methods For this experiment the following materials were used; 4 microtest tubes, 250ml Erlenmeyer Flask, 250ml Beaker, micropipette, 40 µl of reaction buffer, 30 µl of DNA 1, 30 µl of DNA 2, 30 µl of Enzyme 1, 30 µl of Enzyme 2, 20 µl of 10x Gel Loading Solution, .4g of Agarose, ~300 ml of 1x TBE buffer, 2.5 µl of Ethidium Bromide (EtBr), plastic wrap, markers (Standard DNA Fragments), 15 µl DNA from Crime Scene 1, 15 µl DNA from Crime Scene 2, Gel Casting Tray with Gel Comb. Part 1: Preparing the Samples 4 microtest tubes were obtained and, using a new pipette tip each time, reaction buffer, DNA and restriction enzymes were added according to the table below (Upadhyaya, 2017): Table 1 Summary of Additives into Reaction Tubes Reaction Tube Crime Scene Reactio DNA 1 DNA 2 n (µl) (µl) Buffer (µl) Crime Scene DNA with Enzyme 1 Enzyme 1 (µl) Enzyme 2 (µl) X - Samples Suspect 1 Suspect 2 Crime Scene DNA with Enzyme 2 - X 1 2 3 10 10 10 15 15 - 15 15 15 15 - 4 10 - 15 - 15 The DNA from each suspect had equal amounts of reaction buffer and equal amounts of their proper DNA and both were digested separately with equal amounts of the two restriction enzymes, EcoRI and HindIII. Part 2: Casting the Gel To prepare a 50 ml .8% gel, .4g of agarose was mixed with 50 ml of 1x TBE buffer in a 250ml Erlenmeyer Flask. The flask opening was covered with plastic wrap and microwaved for ~1 min. Once the solution cooled, 3.5 µl of Ethidium Bromide (EtBr) was added to the solution (as a stain so when the gel is put under the UV illuminator the DNA will be visible to the human eye), then poured into the gel casting tray with the comb to solidify. Once solid, the tray was removed and positioned so the side with the comb faces the negative pole (the black connection). Then, ~250 ml of 1x TBE buffer was added until the gel was completely submerged and the comb was removed leaving wells in the gel. Part 3: Analyzing the Samples using Gel Electrophoresis The last step to prepare the samples was to add 5 µl of 10x gel loading solution to reaction tubes 1-4 and mix. Then 15 µl of each sample was placed in the following manner (Upadhyaya, 2017): Table 2 Summary of Additives into Gel Lanes Lane 1 Tube Marker What it is DNA Fragments 2 Crime Scene 1 DNA from crime scene with Enzyme 1 3 Crime Scene 2 DNA from crime scene 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 Marker DNA Fragments Once the samples were in the gel, the cover was placed and the electrical wires were connected to their respective colors and the main power source. The gel was ran at 150V/ 150mA for 40 mins. After 40 mins, the gel was removed from the tray and placed on a UV illuminator. The visible bands of DNA fragments left behind from the migrating DNA sample were compared to Table 7.2 and the suspect of the crime was discovered. Results Figure 1. Image of gel showing bands of suspect and crime scene DNA after gel electrophoresis. Analyzing crime scene 1 first, Figure 1 shows that Lane 4, which contained Suspect 1 cut with Enzyme 1, and Lane 6, which contained Suspect 2 cut with Enzyme 1, both very closely resemble similar band separation to that of crime scene 1 that was also cut with Enzyme 1 (Fig.1). The upper bands of Lane 4 and 6 are identical to that of crime scene 1 with the bottom band somewhat fading out. Analyzing crime scene 2 next, Figure 1 shows that Lane 7, which contained Suspect 2 cut with Enzyme 2, is the only sample that shares any resemblance to that of crime scene 2 (Fig 1). Both Lane 7 and crime scene 2 share the similar quality of having a very visible upper band with a somewhat faded lower band. Discussion After observing and interpreting the results of the gel electrophoresis, it was concluded that the suspect guilty of the crime was suspect 2. When cut with the Restriction Enzyme 1, the separation of the bands of the DNA from the crime scene was identical to the band separation of suspect 1 DNA and suspect 2 DNA. Now on the contrary, when cut with the Restriction Enzyme 2, the band distance of the DNA from crime scene only matched that of suspect 2. While the DNA of suspect 2 may be found in the crime scene, despite using two restriction enzymes, this doesn’t make them automatically guilty. Many problems can occur that can invalidate the results or produce inaccurate data. Contamination of the DNA and incomplete ‘cutting’ by the restriction enzymes are two possible factors that may affect the results (Thayer, 1999). There may have been a few errors that could have invalidated the results in this experiment. The first occurred when creating the samples for each reaction tube, it was possible that the amount of DNA and Enzymes added were inconsistent when transferring in the micropipette. Another error that may have transpired was when the samples in the reaction tubes were loaded into the gel wells, if the pipette was not deep enough in the well then some of the samples may have escaped out into the TBE buffer which would make the bands under the UV illuminator hard to see (Laurell, 1966). DNA fingerprinting is an essential tool used in today’s society to determine the guilty or innocent suspects in a crime, such as the experiment done above. In addition to catching criminals, the use of DNA fingerprinting can also be used to perform paternity tests to discover or confirm a childs’ biological father. If only one restriction enzyme was used the results would be inaccurate because there would be less variation in the DNA that could be used to narrow down options. For example, in Figure 1 the crime scene cut with restriction enzyme 1 is almost identical to the DNA sample of suspect 1, cut with restriction enzyme 1, and suspect 2, cut with restriction enzyme 1, this shows that working with only one enzyme does not help make any conclusions (Fig 1).