Dna profiling research paper
Genetic testing adapted from medical and pharmaceutical sciences, such as next-generation DNA sequencing, will soon be applied to mainstream forensic science, opening new avenues in criminal investigations.
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This review aims to cover these key events and advancements in the field from both a historical view and current practice. Keywords: forensic science, DNA profiling, short tandem repeats, mitochondrial DNA, phenotypic testing, next-generation sequencing. DNA profiling has revolutionized the process of forensic human identification. It is of note that this is a relatively recent addition to the forensic science tool kit and that it is constantly undergoing developments.
The aim of this review is to take the reader through the processes of DNA typing, starting with DNA isolation from biological material through novel approaches and phenotypic testing. The review is written for those in forensic medicine; therefore, certain knowledge is assumed, but equally, aspects that are specific to forensic science will be emphasized and placed into the right context.
A Rapid Wire-Based Sampling Method for DNA Profiling
For those who wish more information than can be provided within the scope of this review, there are books ranging from introductory texts 1 to those that are more comprehensive. The first use of DNA fingerprinting was to resolve an immigration dispute in This led to a local police force engaging Jeffreys now Professor Sir Alec Jeffreys to assist with an unsolved sexual assault and murder of two young girls in Leicestershire, England.
DNA fingerprinting led originally to linking the two murders to the same perpetrator, then to the exoneration of a young man, and finally to the conviction of Colin Pitchfork for the crimes in Since , all DNA profiling as it is now termed has centered on microsatellite DNA termed short tandem repeats, or STRs, in this review , and in particular, on repeat units of four bases see chapter 5 from Butler 2 and chapter 6 from Goodwin et al 1.
The STR loci used in standard DNA profiling are all inherited in a Mendelian manner; are not genetically linked, and therefore are inherited independently; and crucially, are thought to have no known association with a disease state. Figure 1 Steps involved in DNA profiling. Profiling starts with isolation of DNA by a process called DNA extraction, followed by quantification of the DNA in the sample, then amplification of short tandem repeat loci, separation of the short tandem repeat products, and finally, interpretation of the genotype data.
Abbreviation: PCR, polymerase chain reaction. The advent and application of polymerase chain reaction PCR 6 to forensic genetics was timely and advantageous. Tiny blood spots or single hairs could generate DNA profiles, thus increasing the sensitivity of the test to subnanogram levels of DNA.
Allied to this were increasing powers of discrimination: the probability that another person shares the same DNA profile by chance. DNA profiling has been heralded as a game changer because of these high powers of discrimination resulting from an increased number of loci being examined between individuals and has been seen to override other forensic evidence for which the powers of association are many orders of magnitude less.
Validation studies and interlaboratory proficiency testing to demonstrate as comprehensively as possible the robustness and reproducibility of the tests were undertaken because of the significance of DNA evidence in any criminal case. DNA frequency databases were generated for all populations to ensure an accurate and frequently conservative estimate of the frequency of the DNA profile.
The chances that the DNA profile came from a close genetic relative were incorporated into statistical formulae, as were mechanisms to adjust the frequencies stated for small subpopulations. The current situation is that because of the extensive validation studies and challenges in courts in many jurisdictions, DNA is now considered a gold standard 7 in forensic science, with the science underpinning the forensic application and processes of evaluation of the evidence being the standard to which other areas of forensic science should aspire.
The forensic DNA community has striven to achieve standardization to ensure the same core set of DNA loci are examined between jurisdictions. This will ensure there is commonality in the data obtained, allowing national and international sharing. This harmonization manifests itself in a number of ways, including, although far from exclusively, the basic steps in forensic DNA processing.
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These are illustrated in Figure 1. Typically, the processes shown in Figure 1 take a minimum of 10 hours, using current standard technology. The aim of much research is to make this process quicker without compromising the quality of the data obtained. Each of these steps is described here. The extraction process chosen may be modified depending on the biological tissue being analyzed. Blood contains DNA only within the white blood cells.
Other cellular components need to be separated and purified from the DNA, such as proteins, lipids, and carbohydrates. Semen has the added complication that the DNA is encased within the spermatozoa head; this head the acrosome is made from a strong protein that is resistant to many naturally occurring enzymes. In contrast, the DNA from buccal and skin cells is more easily obtained, as these epithelial cells are easily lysed, exposing the DNA to the extraction buffer. There are additional considerations for forensic science processing. For example, a forensic laboratory may process tens of thousands of samples every year.
A manual procedure in which individual laboratory staff members pipette small volumes from one tube to another will be highly costly in staff time. Further, every time fluid is passed from one tube to another, it leaves open the potential for contamination or for adding fluid to a wrong or incorrectly labeled tube.
The possibility of using automated extraction processes is therefore beneficial in terms of costs and ensuring that the wrong DNA profile is not attributed to an individual because of a sample mix-up. The principal steps in any extraction process are to lyse the cellular membrane, using a solution typically containing sodium dodecyl sulfate; break open the nuclear envelope, using a solution containing a proteinase typically proteinase K ; separate and purify the DNA molecule ethanol , making use of its negative charge; and elute the DNA into a fresh solution low-salt buffer or water relatively free from inhibitors and of sufficient purity to allow enzymatic amplification of the DNA.
The original method of DNA isolation was an organic extraction process 8 in which, initially, a buffer containing sodium dodecyl sulfate, a chelating agent ethylene diamine tetraacetic acid , and an enzyme to digest protein proteinase K is used to expose the native DNA molecule.
The proteins are removed by addition of an equal volume of the solvent phenol or a mix of phenol and chloroform. Organic extractions are only used in current forensic practice for niche applications, as they are laborious, involve multiple tube changes in this process, and cannot be automated, and because phenol is a highly toxic chemical both to the user and the environment. There are other biological materials that are negatively charged that will coextract with the DNA; hence, this method leaves the DNA dissolved finally in a fluid that is far from free of potential inhibitors.
The silica-based material 10 is embedded in a layer at the base of a spin column, through which fluid containing DNA can pass. The DNA binds to the silica membrane, but all the molecules that do not possess a strong negative charge pass through the membrane and are discarded. The DNA can ultimately be released from its bound state by altering the pH of the silica membrane, allowing DNA to be eluted as a relatively pure solution.poitipincfeara.tk
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Magnetic bead technology 11 acts in a similar way to silica-based methods, in that DNA binds to a biotin complex that is attracted to a streptavidin bound to a magnet. Other constituents of the cell that do not bind to this complex are flushed away or removed.
Once this is complete, the DNA can be released from its binding and eluted into a relatively small volume of fluid. See Table 1 for a comparison of these methods. The earliest method of DNA quantification involved the measurement of the absorbance of light at nm, using an optical density spectrophotometer.
Fundamentals Of DNA Profiling In Forensic Science
Further, this method detects all DNA and is not human-specific. For a while in the s, slot blots were used, in which DNA of known concentration was immobilized on a nylon membrane at specific locations, to act as standards. The DNA samples to be tested were immobilized near the standards. A short sequence of DNA found in humans only actually an alpha satellite repeat sequence on chromosome 17 was used as a probe to bind to the immobilized DNA, and as the probe could be detected by chemiluminescence, the amount of human DNA present could be determined.
This method was more sensitive than using an optical density spectrophotometer to measure the absorbance of light, but it took many hours to complete. The current standard technology used in forensic science laboratories is real-time PCR. As these PCR products are double-stranded molecules, any dye that binds to double-stranded DNA can be used to monitor the reaction as it occurs. Equally, the amount of template DNA present at the start of the reaction can be determined by comparison with known standards.
Using this real-time approach, investigators can monitor a reaction, and if suboptimal nuclear DNA is detected in a sample, they may choose to focus on targeting other markers in the genome that are more ideal for degraded DNA. Real-time PCR has a further advantage, in that the presence of inhibitors can be noted if the reaction fails. The only disadvantage is the cost of the equipment required, as it is significantly higher than the other methods. This can lead to poor amplification, and potentially amplification of the wrong fragments if there is a time delay between preparing the PCR and adding it to the thermal cycler.
The end result is that there is more specific binding in the initial steps of the PCR, leading to more of the targeted PCR product. There has been an evolution in the number of STR loci amplified in one reaction.
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All the STRs used are noncoding, but most are close to a gene sequence or are within introns. The first STR loci were identified within introns and named after the gene with which the intron was associated. For instance, the STR locus called vWA is a repeat found in intron 40 of the von Willebrand Factor gene named after the physician who noted a blood clotting disorder based on a mutation in this gene , which is on the short arm p of chromosome A more simple nomenclature was adopted as more STR loci were incorporated into DNA profiling; for example, STR locus D3S, where D stands for DNA; 3 designates the chromosome; S indicates there is a single copy, rather than multiple copies; and the number indicates that this was the 1,th section of chromosome 3 to be described.
Originally, there were four loci in , 14 , 15 and then six loci 1 year later. By , there were kits that amplified nine STR loci plus a sex test, and then in , a STR locus test was introduced. Another advance in STR technology is that the reaction buffer has seen improvements, such as an increased tolerance to inhibitors. Minor inhibitors of the reaction, which may have previously resulted in no amplification product, have less effect, leading to more complete DNA profiles.