This tubes were ready, they were centrifuged
This practical introduced the students to two of the most basic techniques in DNA analysis: the amplification of DNA fragments by PCR (polymerase chain reaction) and agarose gel electrophoresis (which separates products on the basis of size), by presenting them with a forensics mystery. At a crime scene the DNA (X) of someone other than the victim was found, and now three possible suspects must be tested. To do so, PCR was first performed on the different DNA samples (A, B and C) and a non-DNA control. The products were then separated by gel electrophoresis, along with a DNA marker. Once the specimens were visible, their distance from the wells in which they were pipetted was measured. The same was done for the marker. A graph was then drawn by plotting a marker track first, on which vertical lines from the x-axis, originating from the distances travelled by each band, were extrapolated until they hit the line of best fit. At this point, horizontal lines were drawn to determine the size in kb of the bands. Once the sizes of the specimen were assumed to be correct, a table including both this value in kb and distance travelled in mm, was made. This last step allowed for an easier interpretation of results. As all values calculated for C matched X, it can be concluded that this person is the prime murder suspect. Experimental Design The forensics mystery was solved by conducting DNA fingerprinting. The first part consisted in amplifying the DNA with DNA polymerase. In setting up the polymerase chain reaction, the samples of DNA were first located in ice buckets, as well as the mastermix containing all necessary reagents. PCR tubes were labelled A, B, C, X and control. 24?l of mastermix were pipetted to each and 1?l of DNA sample was added to its corresponding tube, while water for the control. Once the tubes were ready, they were centrifuged and placed into a thermal cycler, which began the actual PCR process. While waiting for the results to be ready, the agarose gel was prepared, following the steps given in the manual, and the gel tank apparatus was set up. By the time the gel was set, the samples and a DNA marker were ready to be loaded and electrophoresis was started, by connecting the apparatus to a power supply of 150V. Once the dye of the samples travelled to about 1-2 cm of the end of the gel, which took approximately 45 minutes, electrophoresis was stopped. Finally, the gel was examined and photographically documented. ResultsI. a) Figure 1 A: Agarose gel electrophoresis of suspects. DM represents DNA marker sample (‘1kb plus DNA ladder’-InVitrogen™ product 10787-018). A, B and C illustrate the DNA samples of the various murder suspects, while X pictures the DNA specimen found on the victim. 0 stands for the water control. 20?l of each sample were added to its corresponding well. The separation was run for approximately 45 minutes, with the apparatus connected to a power supply of 150V. b) Figure 1 B: Class Example. DM represents DNA marker sample (‘1kb plus DNA ladder’-InVitrogen™ product 10787-018). A, B and C illustrate the DNA samples of the various murder suspects, while X pictures the DNA specimen found on the victim. 0 stands for the water control. II. Figure 2: Distance travelled of DNA Samples vs their Size. Based on Figure 1B. As the size of the DNA marker bands was already given, this specimen vs its distance travelled was first plotted. A line of best fit was then drawn. The distance of each band from its well of the various samples was measured in mm, and from these points a straight line was extrapolated until it touched the line of best fit. At this point a horizontal line with a 90o angle was traced to the left, and the size of each band was estimated. On top of each line, its corresponding sample is noted. III. Table 1: Size and distance travelled by bands of various DNA samples SampleDistance Travelled (mm)Size (kp)A 1462.0A 2561.20A 3850.25B 1481.80B 2600.95B 3630.85C 1551.30C 2630.85C 3850.25X 1551.30X 2630.85X 3850.25This table was constructed using the distances travelled measured in Fig. 1B, and the DNA fragments’ sizes estimated from Fig. 2. Each sample is organized from the band that travelled closest to the well to the one that travelled the farthest away. IV. Description of ResultsAs the gel image that resulted after conducting the experiment did not have any visible bands for blood sample A and only two bands for B, the class result image was analyzed instead. The distance of each of the marker’s bands was first measured in mm from its well, and then a graph was plotted using these values vs their corresponding size in kb. A line of best fit was drawn. Once the distance of the bands of all DNA samples was measured in mm, a vertical line from the x-axis was extrapolated to reach the line of best fit, from which a further line was drawn horizontally to the y-axis in order to estimate the size of the bands. Once both values (distance travelled in mm, and band size in kb) was known for each band of each DNA sample, a table was made to allow for an easier interpretation of results. It is noticeable that the values for C and X are the same, making C the prime suspect for the murder. Conclusion The class result was analyzed in this report, as not all bands were clear in the original result; yet, by comparing the two images now, they both have the same prime suspect. This shows that the student was successful on the day of work. The only problem that occurred while conducting the experiment, was not putting in the samples for A and B correctly, and perhaps not enough ?l of A. There are a variety of reasons for which these DNA fragments are not visible. Something may have gone wrong during the PCR, meaning that the DNA bands were not amplified correctly, which would of course result in a not clear gel image. Another possible reason for missing bands could be that not all reagents were added to the mastermix, yet this is very unlikely in this case, as the mastermix was prepared beforehand; if, nevertheless this was the cause for inaccurate results, then it is not the student’s fault as the student did not prepare it. In order to avoid this mistake, or a similar one, the experimenter should make multiple samples of the same substance, to have an extra one in case one doesn’t work correctly. When analyzing the results obtained, Fig. 1B shows that each DNA sample has at least one band that matches the possible murderer’s. Even though it is quite evident that C is the only one that completely matches it, A can be easily confused as well. For this reason it is necessary not only to rely on the gel image but also to make a calibration graph, shown in Figure 2. If bands share just the distance travelled, it is not enough to come to an appropriate conclusion, since many travelled very closely to one another. The marker is important as it provides a ‘track’ on which to measure the other results’ values in kb. Without it, there would be no way to determine the size of all bands. After measuring the distances travelled and drawing the calibration graph to find the sizes in kb of each band, it is noted that C is the only suspect whose DNA perfectly fits the one unknown (X) one. This concludes with a high percentage of certainty that C is the prime suspect.