Polymerase Chain Reaction, Fingerprinting for Crime Detection - Essay Example

PCR based DNA fingerprinting for crime detection Introduction: Invention of Polymerase chain reaction (PCR) by Kery Mullis in 1983(1) has revolutionized the way DNA was looked upon. PCR as name suggests is an enzyme based in vitro technique by which one can generate millions of copy of given DNA in short time. The major constituents of PCR are, 1) DNA polymerase 2) short DNA fragment as primer 3) dNTPs 4) buffer and 5) DNA template. Invention of thermo stable DNA polymerase from thermophilic bacterium Thermus aquaticus (Taq Pol) (2) has made PCR a routine process in all molecular biology laboratories. Currently, there are hundreds of different types of PCR are being used in different laboratories but the basic principle remains same. The overall process of PCR can be summarized as follows. 1) Mixture of all constituents of PCR like dNTPs, Primers (forward and reverse), Buffer and template DNA will be mixed in desired concentration. 2) The first step is amplification, where the temperature is set at 94C for 2-5 minutes for denaturation of double stranded DNA, a process called initial denaturation, 3) After initial denaturation, sample is kept for 30 sec at 94C for further denaturation, 4) After denaturation temperature is brought down to 55-60C for 30sec to allow annealing of primer with specific DNA site called annealing temperature. 5) The temperature is now brought up to the 72C for polymerase to start new DNA synthesis using primer as starting material. 6) After extension, the cycles are repeated for almost 30 times to get 230 copy of initial DNA template. Finally, after 3 cycle extension is be performed at 72C for 5 min to complete any unamplified reaction. Figure 1 shows steps involves in PCR. < http://users.ugent.be/avierstr/principles/pcr.html)> Figure 1: Steps Involves in PCR and exponential amplification Development of PCR and identification of DNA as signature molecules for individual leads to introduction of DNA based technique for establishment of parental relation and subsequently for crime and criminal detection. Based on DNA sequence of humans it was found that there are many places in entire DNA that are conserved nucleotide repeats and based on size or length of these sequences they are termed as micro and mini satellite (4). It was found that number of repeat in these sequences varies from person to person and inherited from parents to child makes it ideal choice for criminal identification. Later, this process was termed as DNA fingerprinting. Moreover, development of PCR made this technique more powerful and realistic compared to any other technique for criminal identification, since most of the time, the specimen obtained in the crime site is always in less quantity. This small part of body or body fluids like blood, sperm, saliva or even hair is sufficient to isolate DNA and then amplification with PCR make it possible to do different analysis on it. Here the aim of this experiment was to understand fundamental principle and use of polymerase chain reaction and based on that to understand how PCR is used in DNA fingerprinting based crime investigation and criminal identification 2. Methods: 2.1 Buccal DNA extraction: Ten ml, 0.9% saline solution was rubbed vigorously against the cheeks for 10 seconds. The sample (extract from the bucaal cavity) was then transferred into 15 ml centrifuge tube and centrifuged at 2000 g for 10 minutes for the pellet. Thereafter, 500 l of chelex beads were added into the pellet and resuspended with the chelex by pipetting in and out various times such that there are no visible clumps of cells. Five hundred microliter of the aliquot was transferred into 1.5ml microfuge tube and was boiled into a hot block at 100C for 10 mins. The sample was then spin for 30 secs top speed to spin down chelex. Fifty microliter of the fresh supernatant was transferred into clean 1.5 ml microfuge tube, taking care that the chelex beads are not transferred and diluted with 450l of sterile ddwater and stored in ice for further use. 2.2. The PCR: Total eight DNA samples were used for the PCR reactions, five DNA samples representing the suspects, one each of crime of scene and the victim. The eighth tube was negative control containing no DNA.PCR mastermix was prepared as follows. 1X reaction Constituent 6.5l Ultrapure water 2.5l dNTPs(2mM) 2.5 l PCR reactions buffer 3.0 l MgCl2 (25mM) 2.5 l Primer 1 DISBOFoo (1 M) 2.5 l Primer 2 DISDORev(1 M) 0.5 l Taq Polymerase 20.0 l Mix volume Twenty microliter of the mastermix was added in the 8 PCR tubes. To each tube, 5l of the each DNA sample was added (except the negative control) and mixed well to give a final reaction volume 25l. Further, 5l of ddwater was added in the control tube. To each tube, a single drop of mineral oil was added to prevent loss of the content due to evaporation, while heating procedure. The samples are ready for the PCR cycles. The Cycling conditions were as followed. 94C, 90 secs Initial denature 94C, 20 secs Denature ds DNA 55C, 30 secs X 25 Anneal primers 72C, 60 secs DNA synthesis 2.3. Gel Electrophoresis: After, the PCR cycles, the product was checked using gel electrophoresis. The ends of the gel tray were sealed with tape and a comb of 1.5cm was placed form the top to create row of wells. The melted agarose was pour steadily into the tray avoiding any air bubbles and was allowed to set for 20-30 mins. Therefore, the combs were removed followed by carefully removing the tape. The tray and the gel were placed in the electrophoresis tank and were filled with TBE running buffer until the gel is submerged. Five microliter of dye was added to each PCR product and mixed well. Fifteen microliter of each sample (dye+PCR product) was loaded into the wells, along with the DNA ladder. The order of the loaded samples and the corresponding samples were noted. The lid was closed and the power supply was turned at 100 volts. The gel was allowed to run, taking care that it does not overrun. 2.4. Staining and Image capturing: The gel was stained with ethidium bromide solution for 20-45 mins. The resultant gel was washed and its image was captured for further analysis. 3. Results: Based on banding pattern it was found that there were three samples or lane having similar banding pattern namely, suspect 2 (lane 2), SoC (lane 6) and victim (lane 7). All three were found to be heterozygous for allele D1S80 and having exactly matching banding. It was also observed that there were two suspects, homozygous for D1S80 locus and hence gave only single band. There was no apparent band observed in negative control lane while molecular markers were uniformly migrated giving molecular weight ladder. 4. Discussion: Band Identity between the samples (scene of crime, victim and suspect 2) clearly indicates that the sample were collected from scene of crime is of either suspect or victims. Matching pattern of victim and suspect may be due to various reasons 1) there might be mixing of sample (in our case the DNA sample in both cases might be of same person), or 2) the criminal might be a close relative of victims like brother or sister. 3) The criminal might have similar repeat for this microsatellite by chance, as the exact matching of minisatellite pattern is probabilistic event. There was no band observed in negative control lane indicating proper PCR without any external contamination To establish criminal 2 as main perpetuator further analysis should be done using other mini satellite marker to obtain distinct banding pattern which distinguish between victim and criminal. In case of multiple allele analysis probability of exact match is almost eliminated due to exponential nature of probability, a reason why in routine crime investigation, investigators use multiple alleles for DNA fingerprinting and identification of criminal to avoid any chance matching and validates results statistically. 5. Bibliography: 1) Specific enzymatic amplification of DNA in vitro: the polymerase chain Reaction Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G. and Erlich, H (1986) Cold Spring Harbor Symposium in Quantitative Biology 51: 263-73 2) Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. Chien A, Edgar DB, Trela JM. J Bacteriol. 1976 Sep; 127(3):1550-7 3) 4) Hyper variable 'minisatellite' regions in human DNA. Alec J.Jeffreys, VictoriaWilson&Swee LayThein Nature 314, 67 - 73 (07 March 1985); Read More