PCR Based DNA Fingerprinting Lab - Research Proposal Example

Introduction In this case study, there is a robbery in property. There were some witnesses who saw four men leaving the scene in a light blue ford focus. The men were in hooded sweat shirts thus their identity could not e established. As per set international investistandards, crime scenes have to be fully swept and examined. When the police officers arrive at the crime scene, they notice a blood stained broken window used for entry the property. Later on, one of the suspects is arrested through via the vehicle they used to escape, the light blue ford focus. The other three suspects are also arrested later. According to the circumstances, the only way to identify the suspects was through DNA profiling using the blood sample from the window as the matching sample. The purpose of this experiment is to establish whether DNA profiling should be used in the determination of criminal cases. The reasons for its used will be based on the accuracy of the DNA test (as will be established in the experiment), ethical, moral, social and legal issues.According to J. Butler (2005), DNA profiling or otherwise referred to as genetic finger printing is a technique through which the identity of individuals is determined by use of their respective DNA profiles. This technique is widely used by forensic scientists in the determination of identity of missing persons, scenes of mass disasters, historical investigations and mostly crime scene investigations. A lot of biological elements can be used in this process including hair, saliva, urine, teeth, tissue, bone, blood, skin e.t.c. Even though, almost all (99.9%) of all DNA sequence in human beings are almost the same, the DNA sample can be used to distinguish every individual unless they are monozygotic twins (J. Butler 2005). These DNA elements are inherited from the parents. In order to differentiate the DNA of individuals, probe subsets of genetic variation are used. These are statistical probabilities of a random match. As such accuracy of the results can be assured. For information from DNA profiling to hold up in any court of law, the DNA typing process must be performed efficiently and reproducibly. Current DNA typing does not consider information that could be similar to various individuals which includes race characteristics such as eye colour and height and pre disposal to disease. As such, the rationale behind the testing and profiling is assured. PCR is a molecular biology used to amplify a short nucleotide sequence across several orders of extent to create a personal DNA fingerprint. Although DNA from different individuals is almost similar, many regions of human chromosomes exhibit a great deal of differences . Such variable sequences are termed polymorphic, with many of these polymorphisms being found in 90% of the human genome that does not code for proteins. Electrophoresis is the motion created by dispersed particles. This is relative to a fluid that is affected by a uniform electric field. PCR amplification uses taq poly­merase enzyme. This enzyme, is stable at very high (near boiling) temperatures. Both the use of PCR and electrophoresis help in the showing the DNA sequence. The experiment is aimed at producing trusted results. Method 1. PCR tubes are labelled. 2. Add 20µl primer mix to each tube 3. 5 µl DNA from extracted samples is added to the corresponding labelled tubes. 4. Each reaction tube is gently mixed using a vortex. 5. Samples placed on ice until all are prepared 6. Sample tubes are then placed into the thermal cycler for a total of 35 cycles as follows. -Initial denaturation : 94⁰C for 3 minutes -35 cycles : 94⁰C for 30 seconds 45⁰C for 30 seconds 72⁰C for 30 seconds -Final extension : 72⁰C for 3 minutes -Once the PCR has completed 5 µl 10x gel loading solution is added to each tube and stored at -20⁰C for unit next session weeks. The gel tank and casting stand with comb are prepared and put in position. 1. 0. 5g of agarose is put into a 250ml flask. 2. The stock buffer is diluted to make 50ml of a 1 x solution. 3. The 1x buffer is added to the agarose in the 250ml flask and the mixture is swirled to mix. 4. The mixture is heated in a microwave on high power for 1 minute 5. The flask is removed with a heat proof glove and swirl the mixture gently, check the agarose has completely dissolved . 6. The agarose solution is poured to the gel casting tray and leave to set. Preparing the gel for electrophoresis 1. Once the gel is completely solidified, the comb is slowly removed from the gel pulling straight up gently to prevent tearing the wells. 2. The electrophoresis chamber is filled with 1x electrophoresis running buffer. 3. The gel is submerged under the buffer before loading samples to the gel. Loading the samples to the gel 1. 200bp DNA ladder and the 5 PCR samples are placed in a hot block set to 50⁰C for 2 minutes. 2. Samples are removed from the hot block and allow samples to cool for a few minutes. 3. The entire volume of each sample tube is loaded to the gel (30 µl) 4. 30 µl DNA ladder is loaded at one end of the gel Running the gel 1. Once the samples have been loaded to the gel and the cover to the gel tank is added making sure the electrodes have the correct orientation. 2. The tank is then to the power source and the correct voltage set. 3. Once the gel has run the gel is removed from the tank for staining. Staining the gel 1. The gel is removed from the tank and placed on some cling film. 2. The gel is then moistened with some electrophoresis running buffer 3. The clear plastic sheet from the Instastain® card and place the unprinted side of the card onto the top of the gel. The card is the pressed the card onto the gel firmly. 4. The setup is then allowed to stain for 10-15 minutes. 5. The Instastain® card is then removed to view the gel on a UV transillumminator. Results The resulting image is as follows. In this case, the identification of the suspect is possible. This is from the observation of the alleles sequence and patterns. The DNA sample from the crime scene matches that of 4 sample (RE01) which belongs to Russell Eves. This conclusion is made from the careful observation of the alleles. The other three samples do not have a match to the sample from the crime scene. This is because of the lacking the element of double strands. One has to view multiple STR loci simultaneously. From this point, a difference can be noted in the allele sequence and as such identification made. The pattern of alleles being viewed can identify any individual with quite a high degree of accuracy. As such STR analysis usually provides an excellent tool for identification (J. Butler 2005). Discussion A 200bp DNA Ladder is used to determining the size of double-stranded DNA. This is from 200 to 3,000 base pairs. It consists of eleven fragments which have different ranges in size. Its main function is to set the standardization and comparison table for the test. Over time, there has been a debate of issues surrounding DNA profiling. These issues range from social, legal and ethical issues. In various countries, acts of parliament, congress and senate are being put in place to handle the various ethical, social and legal issues surrounding how the DNA typing process is conducted, who conducts the process and to what use is the gathered information put into. The values in which the above issues are concerned with are primarily in liberty, privacy, autonomy, informed consent and racial equality. One of the most pressing issues is genetic privacy. Once the DNA of an individual is taken and uploaded in the data base, it will stay there until the individual applies for removal. This process is tedious and time consuming. As such, many people would not want to get involved with it. The DNA is available for viewing by the security agencies and therefore, no privacy is affected. The DNA can also be matched with that of family and relatives. This is violation of privacy. The mode through which the DNA samples are obtained is also to questions. In case the mod of acquiring the DNA sample is without the consent of the individual, then the privacy has been violated. The use of the DNA samples that are stored in the data base is also a violation of privacy. However, the security agencies justify this by saying that national security is far much esteemed than the privacy of an individual. DNA profiling has been in existence for a number of decades now. As much as it has helped solve a lot of crimes, it has also brought about several legal social and ethical issues along with it. These issues include interference with individual privacy, discrimination and security. The results of the process have been obtained through PCR. Over the last few years, almost all research conducted on human DNA quantitation has had its focus on new and real-time quantitative PCR techniques. Quantitative PCR methods have enable precise, automated, and high-throughput measurements. For this reason, the use of DNA typing is rationalized. PRC will give faster results in the identification and thus help solve crimes and other societal issues. From the case study, the criminal is identified through the DNA match. References. J. Butler (2005), ‘Forensic DNA typing’, Elseveir. A. J. F. Griffiths, S.R. Wessler, R. Lewontin, & S. B. Carroll, ‘Introduction to Genetic Analysis’ 9th Ed., 2007, W.H. Freeman. R. E. Gaensslen, H. A. Harris & H. Lee (2008), ’Introduction to Forensic science and Criminalistics’ McGraw-Hill. I. Pepper ‘Crime Scene Investigation: Methods and Procedures’, 2005, Open University Press Berg J. M, J. L. Tymoczko & L. Stryer ‘Biochemistry’ 6th Ed, 2006, W.H. Freeman. Read More