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Forensic Identification of Criminal with use of PCR (Polymerase Chain Reaction) 1.

1.1. Polymerase Chain Reaction - PCR The polymerase chain reaction (PCR) is a technique that had great impact on development of many scientific techniques in molecular biology. Relatively simple method improved gene technology and analysis by amplification of diminutive DNA sequences producing thousands to millions of DNA amplified copies. Polymerase chain reaction became indispensable technique which facilitated many diagnostic applications such as identification in forensic science through minimum of DNA present (e.g. daktyloscopy, small samples of blood, semen, hair root), also pre natal diagnosis derived from foeatal tissue, DNA cloning and analysis of hereditary diseases. Principles Basic PCR is based on thermal cycles with alteration of heating and cooling reactions. Requirements for successful PCR run involves presence of two oligonucleotide primers situated at the 3 end of the DNA strand, DNA template, DNA polymerase and PCR buffer containing Mg2+ ions. PCR technique applies three major thermal cycles which are responsible for, in order: denaturation, annealing, and elongation of the DNA sequence required. PCR cycle is usually repeated 30 35 times where each cycle consists of variation of temperatures with individual steps of the PCR. The first part of PCR cycle is denaturation (approx. 90-950C for 20-30 seconds) which causes DNA template strand to melt, thus disrupt the hydrogen bonds between the complementary bases. At this stage, due to high temperature primers are not able to bind and re-form the hydrogen bonds. In the second step of annealing, now two individual DNA strands can due to lowered temperature create bond with complementary primers on the 5side, after which elongation can occur. Annealing occurs at approximate temperature of 50 to 550C for next 20 seconds. Throughout the final step, the temperature is raised to 720C for 30 seconds in the crucial presence of DNA polymerase (Taq polymerase) and nucleotides (dATP, dCTP, dGTP, dTTP). DNA polymerase cannot recognize the end of the target sequence, hence it synthesize new DNA until the next cycle denaturation when temperature automatically increases. The new hydrogen bonds (in 5 to 3 direction) can be produced between added nucleotides and complementary DNA template bases. In the second cycle, primers repeatedly anneal to the template DNA but also to the newly synthesized DNA strand. This process repeats approximately 30 times. To make sure that DNAs are fully synthesized the additional 10 minutes can be added at the end of the whole PCR cycle. For the final results agarose gel electrophoresis is most common technique applied and will be discussed in next section of the introductory part. As shown in the Figure 2.1- DNA Amplification Using Polymerase Chain Reaction, the DNA strand is shortening exponentially with repeating cycles. 1.2. Arbitrary primers- DNA identification by arbitrary primer PCR Forensic analysis has developed a versatile method for identification of various DNA samples such as fingerprint by applying arbitrary primers. Arbitrary primers are selected primers that have the ability to initiate the DNA synthesis even the primers do not match perfectly; hence no sequence information is required. 1|Page

1.3. PCR Troubleshooting The amount of target sequence to be amplified may result in no band produced during electrophoresis on agarose gel. Large amount of the DNA picked from the colonies could result in no band production, thus leading to mis-priming and also poor DNA synthesis due to affected diffusion of the large polymerase molecule (e.g. Taq polymerase). In contrast, small amount of DNA picked from colonies could result in its complete loss (e.g. chemical degradation, enzymatic degradation). It also increases the risk of contamination literally from anything that DNA could come across. Diminutive amount of skin, exhalation, or hair root could result in degrading the DNA to be tested as they can carry both the DNA and the degrading enzymes such as nucleases. These all could affect the results presented in the Figure 3.2 where sample taken from bacterial colony did not produce any band as well as the unknown sample A did not appear on agarose gel. Furthermore, inadequate concentration of dNTPs where excessive amount would inhibit the production formation or even prevents PCR from working. Primer increased concentration could also inhibit the product formation by forming dimmers or leading to nonspecific product. Polymerase concentration has also great impact on the PCR as small deviations may result in incomplete primer elongation as well as premature termination. All factors listed may have great impact on PCR and delivering the product which can be recorded from the agarose gel.

2. Aim
The major objective of this experiment is valuable identification of the unknown delinquent who sabotaged the Cup Final game by poisoning the food of very important player of the Fulchester Willy the Whale Thomlins. In order to identify the criminal, samples from the food Willy Thomlins ate were taken and will be analysed with PCR and separated on agarose gel.


3. Results and Discussion

Figure 3.1 Control Experiment Gel with Samples from Bacterial Colonies as well as the Unknown Scenarios Samples

Figure 3.1 demonstrate control gel where three lines X, Y and Z represents the samples obtained from bacterial culture, whereas samples A, B and C represent the pure plasmid samples with unknown identity Figure 3.2 PCR Results Figure 3.2 demonstrates the PCR results obtained from the three various colonies marked as X, Y and Z and the unknown sample A. Three samples demonstrated bands produced by all three options hence the sandwich prepared by victims mum, sample from drink served by barman and energy bars given by Doctor. The unknown sample A taken from the victim was expected to produce the band matching the size of one of the samples X, Y and Z. However, this was not the case as sample X did not produce any band and also unknown sample did not exhibit a band. Consequently several factors were considered in section 1.3 that could have affected the PCR and thus the production of bands on agarose gel. Because the unknown sample was not taken from the cultured bacterial colony, it was thought to be contaminated with a foreign DNA. Foreign DNA could come from debris of the analyst as well as


simple exhalation could affect the DNA sample

and misrepresent the results

Table 3.1 Agarose Gel Readings from Bacterial Colony Samples X, Y and Z Colony from Migration Distance (mm) Size of Fragment bases Log [size of fragments] X 40.5 700 2.845 Y 45 600 2.778 Z 38 800 2.90

Table 3.2 Agarose Gel Readings from Bacterial Colony Samples A, B and C Unknown Sample Migration Distance Size of Fragment Bases Log [size of fragment] A 46 572.796 2.758 B 41 704.693 2.848 C 38.5 781.627 2.893

Graph 3.1 DNA Calibration Curve

DNA Calibration Curve

2.92 2.9 Log DNA Size [Bases] 2.88 2.86 2.84 2.82 2.8 2.78 2.76 36 38 40 y = -0.0182x + 3.5864 42 44 2.77 46 Linear (DNA Calibration Curve) 2.84 DNA Calibration Curve 2.9

Migration Distance (mm)

Figure 3.3 Fragment Sizes Calculation for Unknown Sample A, B and C According to Calibration Curve Equation Log [y A] = - 0.018x + 3.586 Log [y A] = - 0.828 + 3.586 Log [y A] = 2.758 Antilog (y A) = 572.796 Log [y B] = - 0.018x + 3.586 Log [y B] = - 0.738 + 3.586 Log [y B] = 2.848 Antilog (y B) =704.693 Log [y C] = -0.018x + 3.586 Log [y C] = - 0.693 + 3.586 Log [y C] = 2.893 Antilog (y C) = 781.627


Graph 3.2 DNA Calibration Curve with Unknown Samples A, B and C

DNA Calibration Curve

2.92 2.9 Log DNA Size [Bases] 2.88 2.86 2.84 2.82 2.8 2.78 2.76 2.74 0 10 20 30 40 50 Migration Distance (mm) y = -0.0182x + 3.5864 C 2.77 2.84 A B 2.9 Series1

4. Conclusion
Despite the attempted identification of the unknown criminal responsible for the food poisoning of important football player, the PCR did not produce sufficient results. The speculations are concentrating around the contamination by possible DNA as well as possible digestion by nucleases present of the debris, hair root and saliva from exhalation. For further identification the test would need to be repeated.