Browsing by Subject "technology"
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Item Amplified Fragment Length Polymorphism Analysis of White Oak Tree Leaves(2005-07-01) Patel, Kaajal Devendra; John Planz; Joseph Warren; Arthur EisenbergThe AFLP technique at first seems to be a remarkable new technology that can be applied to the growing area of non-human DNA testing. The ability to identify organisms without prior genetic knowledge would be an asset to a field such as non-human DNA testing since not enough research in the area is being conducted. With any new technique or theory in science, intense scrutiny must be used to examine the applicability of the new technology. In the area of forensic science, the severe consequences of a false result extend far beyond the realm of scientific error. Errors make in forensic casework could result in life changing occurrences for the families of not only the victim, but the defendant as well. From this study it can be seen that AFLP as a technique may not stand up to the high expectations of reliability, and reproducibility required for a technique to be adopted into the field of forensic science. Several problems occurred through this study that may prevent this technology from becoming a widely accepted technique in non-human DNA testing. The initial problems with the technique were associated with reproducible results. The first several attempts were conducted under the same conditions, by the same analyst but yielded results that were no comparable. The RFUs of each experiment were inconsistent, not only between samples examined at different times, but samples examined within the same tiral as well. AFLP as a technique is supposedly insensitive to template concentrations however, it has been previously shown to produce differences in the electropherogram when the template is excessively diluted (26). Vos et al. (1995) determined that high dilutions yielding template DNA concentrations below 1 pg could result in irreproducible fingerprints. In this study 27.5 ng of template DNA was added to each digestion-ligation reaction, yet the resulting quantity of amplified fragments varied. These variations in quantities of amplified product could be due to PCR inefficiencies when comparing samples from different trials, but it does not explain instances where duplicate trials were inconsistent with each other (10, 22). When new ligase was introduced the resulting electropherograms did produce considerably higher RFUs for each peak, but the lack of interpretable peaks observed previously may not have been solely due to inefficient ligase. In an inter-laboratory study, Jones et al. (1997) noted that several laboratories encountered problems in obtaining complete AFLP profiles. For several groups, up to 50% of the bands were missing during the preliminary testing. Though this problem subsided with successive attempts, this approach to achieving successful results may not be feasible in a forensic setting. Often the evidence received from a crime scene may be insufficient to allow for multiple testing. In addition, multiple attempts to obtain results may open up areas for scrutiny and attack by the defense counsel. Repetitive testing may appear to be a biased search for condemning evidence against the questioned party, rather than the production of reliable results. Repetitive testing may also not be possible since laboratory reagents and time involved in the production of these results may not be within the constraints of a crime laboratory. In this study, capillary electrophoresis was used to visualize the fluorescent dyes attached to each fragment however, laboratories could use radioisotopes and polyacrylamide gels instead. This method of visualizing AFLP fingerprints is not only costly, but time consuming as well. Conducting repetitive tests in order to obtain a sample with sufficiently intense bands for analysis may not be feasible. These limitations may therefore restricts the use of the AFLP technique from only being conducted in laboratories with sufficient time and funds to conduct repetitive testing as is needed (10). Despite the potential cost in time and funds, the technique was able to produce AFLP fingerprints that were consistent with each other when the electropherograms were compared. The major source of error resulted from the method used to determine the presence of peaks within the designated categories. Since not all peaks crossed the 50 RFU detection threshold, they were not identified by the Genotyper macros. However, when the actual electropherograms were compared, these peaks were present. It has been suggested that to verify whether each peak is present in the pre-designated categories a scan of the electropherogram should be done and any peaks that were not called by the macro should be manually entered into the binary table or should be reanalyzed (Heather Coyle, personal communications). Although this method could potentially aid in the correct genotyping of each sample, it requires a considerable amount of user intervention. A considerable amount of time is needed to examine each electropherogram for the presence of peaks that are below the 50 RFU threshold. Without a redefined interpretation threshold, the analysis of each electropherogram can be highly subjective. Peaks that are relatively low need to be distinguished from peaks that may be associated with background noise. Therefore, in order to eliminate analyst bias a peak detection threshold must be established. Generally the interpretation threshold is established by a validation study of the analysis technique. In this study the lower threshold was previously established at 50 RFU for the instrument being used, but this threshold was insufficient for the recognition of all peaks present during the AFLP analysis. The question then becomes to what extend the peaks can or should be called in order to correctly identify each organism without errors. The exclusion of some peaks could lead to discrepancies, such as those observed during the blind study, which could result in an initial false match or exclusion. The interlaboratory study by Jones et al. found only one scoring difference associated with the absence of one band out of a total of 172 in the AFLP profiles. This error was later associated with experimental errors that incurred during the AFLP procedure. Discrepancies such as this can lead to an erroneous identification of samples that could have severe consequences in a criminal case. At this time, the utilization of AFLP technique for further testing of other organisms such as Cannabis sativa does not seem feasible. A variety of adjustments in the technique need to be addressed before this technology should be further applied to organisms in forensic casework. In order for AFLP typing to be used for forensic casework, major improvements in the technique need to be made. Consistency in obtaining reliable electropherograms with peaks well above the RFU detection threshold must be resolved in order to allow for accurate sample interpretation. This will not only allow for greater consistency between replicates, but will also help in establishing new databases for organisms that are being tested. As with any type of forensic DNA analysis, a database must be established for each organism being tested. Without a reliable database, accurate identification of crime scene evidence cannot be established. A major improvement that is required for the utilization of AFLP typing is the process by which genotypes are identified. Utilizing the macros to identify control and variable peaks to create the binary table was a quick and easy method, however it was not always able to identify the correct genotype. The overlapping of electropherograms in GeneScan ultimately was the best method for accurate identification of the blind samples, but in a real case scenario it would not be feasible to compare each evidentiary electropherogram with those in a database. Advancements in technology will continually introduce new techniques and procedures that could be applicable to the field of forensic science. As with any new technique, the methods and theories must be validated in order to determine whether they can be used in a criminal case. The field of non-human DNA testing is growing and with the advent of new technology such as AFLP, the possibility for establishing a non-human DNA identification method may be on the horizon.Item Evaluation of Y-STR Data Using a Duplex Gender Real-Time PCR Assay on an ABI Prism 7000 SDS Followed by Amplification with Applied Biosystems AmpFLSTR Yfiler PCR Amplification Kit(2007-08-01) Miller, Jennifer J.; John Planz; Arthur Eisenberg; Joseph WarrenQuantification is the process of determining the concentration of DNA in a sample and plays an extremely important role in the processes of amplification and STR typing. A method of quantification is mandated for a laboratory conducting forensic DNA analysis by National Standard 9.3 (1). Furthermore, anytime a forensic laboratory chooses to implement a new or novel methodology for any step in DNA analysis, a laboratory must conduct an internal validation to ensure the quality of method and any results generated on the equipment used within that laboratory are reliable, reproducible, and accurate before the method is utilized for casework analysis (1). Prior to an internal validation, the method or technology must undergo a developmental validation by the developer or manufacturer to determine conditions or limitations of the method or technology on DNA analysis of forensic samples (2). A study has shown that Y-STR results can be obtained even when the quantification of samples yields a value of 0.00ng/μl (4). The issue of the absolute lowest limit of detection in the quantification process versus input DNA concentrations of the unknown samples to yield any valuable Y-STR typing data has not been addressed. A duplex gender assay developed by Nicklas and Buel (3) has a reported detection limit of 0.5pg for the Alu probe of the duplex assay and quantification will be evaluated on a different qPCR platform than originally reported and followed by amplification using Applied Biosystems’ AmpFLSTR Yfiler PCR Amplification Kit to assess quantification limits. The goal of this internship project was to complete a preliminary evaluation of the sensitivity of a quantification methodology on a different qPCR platform under different detection parameters utilizing Y-chromosome DNA in correlation to Y-STR typing results and evaluate the data qualitatively.Item Remote Recruitment and Data Collection and Its Effect on Demographics and Outcome Variable Scores(2019-12) Cooper, Savannah B.; Dory, Ladislav; Licciardone, John C.; Gwirtz, Patricia A.; Kearns, CathleenBACKGROUND: Technology continues to push different aspects of research forward, including new recruitment methods and updated data collection/management. More and more, researchers are beginning to use social media as a valid recruitment method. Studies are also moving toward more remote methods of consenting research subjects and data collection. Several studies were found to use only online recruitment methods and many showed that Facebook was an effective method [1]. With this shift toward more remote and more technologically advanced research, it is important to explore whether increasingly adding technology to research studies shifts the study population in a way that impacts outcome measures. Based on previous research, studies with mainly remote modes of recruitment and data collection have a younger, more educated and less diverse population [2] [3] [4]. HYPOTHESIS: (1) By relying on technology and fewer interpersonal interactions, the demographics of the study population will shift toward younger participants, increased employment status, higher education level, fewer minority participants, and gender will shift to a more even balance of males to females. (2) Changes in the population demographics that are driven by technology will also increase the SPADE cluster score (Sleep disturbance, pain, anxiety, depression, and low energy/fatigue). Specific Aims for both Hypothesis I and II include; (1)To examine the demographics of the study population and outcome variable scores when looking at technology-focused recruitment methods implemented at different points in the study. (2) To examine the difference in demographics and outcome variable scores when looking at effects of the recruitment method alone (without the time of enrollment consideration). METHODS: Survey data from the baseline visit of 583 subjects in the PRECISON Pain Research Registry were used. The subjects were divided into groups based on changes in recruitment methods over the course of the study to evaluate how changes in primary recruitment methods may have shifted the population. The same 583 subjects were also divided into two groups: traditional methods and online methods to evaluate the effect of recruitment methods alone on the population. Demographics and the SPADE score outcome variables were analyzed using chi-squared and t-test analysis to see if there was a significant change between the groups. RESULTS: For Aim 1, there were no statistically significant changes in the population demographics or the SPADE outcome measures with the only exception being gender. For gender, the proportion of females has significantly increased as the reliance on digital methods, such as online newsletters and social media, has increased. For Aim 2, comparing enrolled subjects who were recruited through traditional methods such as flyers in clinics and the community to subjects who were recruited through online vehicles regardless of when they enrolled in the study, there was a significant difference in every demographic except ethnicity. The online group showed a significantly younger, more educated, and less diverse population. DISCUSSION: Hypothesis I was not supported by Aim 1 but was supported by Aim 2. This means that over the course of the study time the population has not changed dramatically, possibly because a mix of traditional and online recruitment methods is still being used. However, the population that was recruited using online methods was significantly different than the population recruited through traditional methods. It is important to keep this in mind as the study moves forward. Hypothesis II was not supported by either aim. Aim 1 showed minimal changes and Aim 2 showed a trend rejecting this hypothesis. Currently, there is no data to support that technological advances would have an increase the outcome variable scores.Item Secondary Science Curriculum: Developing a Browser-Based Constructive Ecosystem(2004-08-01) Barrier, Ame R.; Reeves, Rustin; Shores, Jay H.; Rudick, Victoria L.Barrier, Ame R., Secondary Science Curriculum: Developing a Browser-Based Constructivist Ecosystem. Master of Science Education (Biomedical Sciences), August, 2004, 46pp, 1 software program, bibliography, 33 titles. The internship practicum report explores current research on the plausibility of incorporating technology based on guided inquiry into K-12 classrooms to increase student science achievement. Part of the practicum report is browser-based software designed for teaching an ecosystems unit and includes the materials for use in a guided inquiry classroom. Can browser-based science instruction designed along a guided inquiry approach increase student achievement in science education and be quantified when compared to didactic classroom methods? The practicum report and the materials developed are designed to provide a means for the eventual testing of technology infused guided inquiry against traditional didactic teaching in the K-12 science classroom and begin to answer this question.