PREPARED BY: Iciar Roldan AresABSTRACTThis practical provided a murder scenario in which, using the technique of DNA fingerprinting, three suspects (A, B and C) were studied to match the traces of blood found on the victim. During this practical the use of the PCR (polymerase chain reaction) and the separation of DNA products by agarose gel electrophoresis was required to provide a crucial clue to the murderer’s identity. As a result, the DNA from suspect C coincided with the DNA from the victim’s body, evidence which was needed for the prosecution.MATERIALS & METHODSIn setting up the PCR, five samples of DNA are studied: label ‘X’ for the DNA from the crime scene, label ‘A’, label ‘B’ and label ‘C’ for each of the three suspects and label ‘No DNA control’ for the control sample. 24ml of ‘mastermix’ was added into the bottom of five PCR tubes, followed by 1ml of each DNA sample from each sample tube containing DNA and 1ml water (‘no DNA’) was poured into the control tube. Once mixed, the lid of five Eppendorf tubes was cut to introduce the smaller PCR tubes inside, making sure the lid of these last tubes was closed. Again, these were span for a few seconds to gather the liquid contents to the bottom of each tube. The uncapped PCR tubes in the PCR machine (thermal cycler) were incubated for 45 minutes. While in the PCR machine, the flatbed agarose gel was prepared to analyse the DNA products, done by checking the gel tank apparatus and developing the gel solution (0.5ml of 0.5´ tris-borate-EDTA (TBE) buffer was introduced into a tube of SybrSafe DNA dye and mixed carefully). When accomplished, agarose was dissolved by heating. During the cooling of the agarose, the gel tank apparatus pieces were placed correctly and the joins were sealed with the agarose solution. Finally, the gel was poured into the apparatus. When the gel was set, ICE-COLD 0.5´ TBE buffer was introduced into the gel tank, the marker sample and the DNA samples were inserted into each well too. Once the gel tank was turned on, the electrophoresis was achieved, so the results were obtained.RESULTS i Figure 1A- Agarose gel electrophoresis from suspects and murderer’s victim. Label ‘0’ for the ‘No DNA’ sample, label ‘X’ for the murderer’s victim, label ‘C’ for sample C, label ‘B’ for sample B, label ‘A’ for sample A, label ‘DM’ for DNA marker.i Figure 1B- Guide agarose gel electrophoresis, class example. Label ‘0’ for the ‘No DNA’ sample, label ‘X’ for the murderer’s victim, label ‘C’ for sample C, label ‘B’ for sample B, label ‘A’ for sample A, label ‘DM’ for DNA marker.ii Figure 2- Graph of the agarose gel electrophoresis. Log size DNA fragments plotted against distance migrated on the gel for each band in the ‘marker’ track. The line drawn between the points on this graph is the line of best fit from the traces left by the ‘DM’ marker. Sizes of samples ‘A’, ‘B’, ‘C’ and ‘X’ are estimated using this line, measuring the trace of each sample in mm, it is placed on the x-axis to match the line where it can be estimated the size of each base pair from the y-axis.SAMPLEDISTANCE TRAVELLED (mm)SIZE OF BAND (bp)A1351350A240900A358210B1341450B243700C140900C244650C358210X140900X244650X358210iii Table 1- Samples results regarding the distance travelled (mm) and sizes of the bands (bp). Results collected from the combination of figure 1A and figure 2. Figure 1A was used for this table considering the results obtained were clear. Therefore, introducing the distances from the samples A, B, C and X (figure 1A) in the graph (figure 2), the base pair size was calculated. It was found in table 1 that sample X (murderer’s victim) and sample C had the same base pair sizes (C1 and X1- 900bp, C2 and X2- 650bp, C3 and X3- 210bp) and the same distance travelled (C1 and X1- 40mm, C2 and X2- 44mm, C3 and X3- 58mm).DISCUSSIONThe results of the practical suggested that the blood found under the fingernails of the victim matched sample C. Examining samples A, B, C and X, differences existed in the traces from samples A and B, however, C and X appeared to be the same. This could indicate that sample C and sample X could be the same person, as sample C had the same base pair sizes (C1 and X1- 900bp, C2 and X2- 650bp, C3 and X3- 210bp) and the same distance travelled (C1 and X1- 40mm, C2 and X2- 44mm, C3 and X3- 58mm). Although in Figure A1 only two traces could be detected from sample B, it could be excluded as sample B1 did not match sample X1 (B1 base pair size 1450bp and X1 base pair size 900bp, B1 distance travelled 34mm and X1 distance travelled 40mm). These last numbers were provided by table 1. The practical was followed with extreme precautions so that DNA samples were not contaminated throughout the experiment and the reliability of the results was not altered. Overall, it was certain to say that the victim’s murderer (sample X) coincides with sample C. Therefore, sample C was the prime suspect.