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Item Advanced Problem Solving in the Biotherapeutics Industry: Parameters influencing the delivery of a novel cell therapy product and exploration of a new method for determining activity of Clostridium histolyticum collagenase, a wound debridement enzyme(2015-05-01) Harris, Melanie A.; Jerry W. Simecka; Patricia A. GwirtzBiotechnology is a multi-faceted industry with many unique challenges that require knowledge in a broad range of topics. When working in the wound care field it is necessary to not only create a product in the laboratory, but also effectively bring it to the patient. This task requires many skilled people who can test it for efficacy, design and conduct clinical trials, confirm quality and consistency, design packaging, consider transportation issues and so on. The following investigation focuses on the testing of a cellular product and its accompanying device under various conditions as well as the exploration of a new assay capable of the activity of a wound debridement enzyme. The results of the product/device testing have generally confirmed the comparability of the cellular product devices as well as their resistance to various temperatures encountered in the clinical environment. A new modified assay for the testing of collagenase has been established as precise and comparable to current methods, though it requires more testing to confirm robustness.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 An Initial Comparison of Applied Biosystems Quantifiler Duo and Promega Plexor HY Real-time PCR DNA Quantification Systems(2008-05-01) Cole, Sarah Kathleen; Arthur Eisenberg; John Planz; Joseph WarrenObjective 1: Sensitive Study: This study was designed to determine the quantity of template DNA below which amplification is not expected to yield a DNA profile. Dilution series of male and female stock DNA ranging from 0.003 ng/μl will independently be run with both Quantifiler Duo and Plexor HY. These samples will be run in duplicate per plate, with duplicate plates being run. We want to determine if the published lowest detection thresholds (0.023 ng/μl for Duo; 0.0032 ng/μl for HY) are concordant with the data obtained. Objective 2: Mixture Study: The purpose of this study is to obtain quantification results for mixtures of male and female DNA, which should allow for calculations of autosomal:Y ratios that can be helpful in determining what type of genetic analysis to pursue (autosomal STR, Y-STR, or both). Mixtures of female and male DNA ranging from 1:1 to 1024:1 (female: male) will be run in duplicate per plate, with duplicate plates being run. We want to find out how minor of a contributor the male can be in an excess of female DNA and still be detected. This is especially important in sexual assault cases where the major contributor is usually female or when the offender is a vasectomized male. Objective 3: Concordance Study: The purpose of this study is to compare quantification results from Quantifiler Duo and Plexor HY with those from Quantifiler Human, specifically in cases when samples are degraded. The majority of these samples originate from unidentified human remains. Patterns of overestimation or underestimation of DNA concentration can help determine which system will be most beneficial in these cases. This is where the new amplicons size featured in Quantifiler Duo is important in comparing the values with previous results for Quantifiler Human. Sample choice will be at the discretion of the laboratory technical leader and Unidentified Human Remains section analysts. These samples will be the ones that are known to be degraded and have previously yielded overestimated results from the Quantifiler Human quantification system, resulting in poor STR data.Item Analysis of Low Copy Number DNA Using Profiler Plus at Increased Amplification Cycles and Modifications in Sample Injection Parameters(2003-08-01) Hynds, Jody Lynn; Arthur Eisenberg; John Planz; Joseph WarrenThere are many DNA testing techniques that can be utilized for samples with low quantities of DNA. Mitochondrial DNA testing is designed for successful DNA sequencing of hair shafts, degraded and burned samples. Newly developed SNP (single nucleotide polymorphisms) testing is also designed for the analysis of challenging samples. The increased interest in the analysis of low copy number DNA samples using STR testing is necessitated since the national database CODIS (Combined Data Index System) currently only accepts the DNA profiles analyzed with the 13 core STR loci. CODIS contains DNA profiles of evidence found at crime scenes, convicted offender and missing persons DNA profiles (4). The goal of this project is to develop methodologies to increase the success rate of LCN DNA samples using STR testing. The experimental design for this study involved the amplification of DNA isolated from buccal swabs using the Profiler Plus multiplex kit at two different DNA input quantities: 0/0156ng (15.6pg) and 0.0312ng (31.2pg). Four separate amplifications of these DNA samples were done at: 28, 30, 32 and 34 cycles. The manufacturer’s recommended cycle number for AmpFISTR Profiler Plus is 28 cycles. These samples were analyzed on both the ABI Prism 310 Genetic Analyzer and the ABI Prism 3100 Genetic Analyzer using OCD standard protocols for loading samples. The injection time and voltage were modified for each of the number of PCR cycles. The best combination of cycle number and injection parameters was chosen for the low copy number reproducibility study.Item Automatable Virtual Array Screening System for Rapid Analysis of Mitochondrial DNA Polymorphism(2002-05-01) Campbell, Rowan Stewart; Arthur J. Eisenberg; Bruce Budowle; John PlanzCampbell, Rowan Stewart, Automatable Virtual Array Screening System For Rapid Analysis of Mitochondrial DNA Polymorphism. Doctor of Philosophy (Biomedical Sciences), May, 2002, 156 pp., 11 tables, 48 illustrations, bibliography, 96 titles. The goal of this research project was to develop alternative methods to traditional forensic mtDNA sequence analysis. Conventional forensic mtDNA analysis requires the direct sequencing of Hypervariable Region I and Hypervariable Region II in both the forward and reverse directions. This method is time consuming, labor intensive and expensive. Two methods for determining mtDNA haplotypes through the direct interrogation of Single Nucleotide Polymorphisms with HVI and HVII have been developed. A Sequence Specific Oligonucleotide Hybridization assay was developed on the Luminex 100™ flow cytometer, as well as a Single Base Extension assay developed for the ABI Prism® 310 Genetic Analyzer. The SNP typing of mtDNA sequences can provide a significant benefit in many forensic and human identification cases. The reassociation of mass disaster remains, mass grave analysis, and the screening of large numbers of crime scene samples are examples of their potential application. Their inclusion as a standard screening tool would be high beneficial since more extensive DNA analysis would be reserved for those samples that possess the greatest evidentiary value. In a blind study of 50 samples, the Sequence Specific Oligonucleotide Hybridization assay incorrectly identified the mtDNA haplotypes in 7 samples, whereas the Single Base Extension assay correctly identified each of the SNP positions interrogated. The SNaPshot™ primer extension assay was approximately 20-25 times more sensitive than the standard sequencing approach. This would suggest that this system could be a viable alternative to sequence analysis when samples are limited, as well as being more robust in detection and typing of heteroplasmic sites. A statistical evaluation of the SNP panels revealed that the genetic diversity estimated for the 50 Southwestern Hispanic samples tested was 0.9624 for the primer extension array and 0.9559 for the hybridization-based array. The probability of two randomly selected individuals from a population group having the same mtDNA haplotype was 0.0568 for the Single Base Extension assay and 0.0632 for the Sequence Specific Oligonucleotide Hybridization assay. A forensic mtDNA SNP array consisting of the positions evaluated in this study could provide a reasonable alternative to the full sequencing of the HVI and HVII regions.Item Beta Testing and the Population Genetics of Promega's Prototype PowerPlex Y Kit(2004-08-01) Kirkendoll, Ross A.; Joseph Warren; John Planz; Arthur EisenbergDevelopmental validation is typically done by the manufacturer of the technique or technology. According to National Standards, the manufacturer must test for human specificity to ensure compliance with standards. In addition, the PowerPlex Y kit must be shown to have male specificity because all of the loci are located on the Y-chromosome. Other necessary studies include mixture both male/female and male/male mixture studies, stability studies to show stability in the presence of environmental insults, and the focus of this study the construction of a popular database. In order to satisfy both the requirement of the National Standards and the scrutiny of the legal system, Promega Corporation assembled a collaboration of different laboratories to assist with the developmental validation of the PowerPlex Y Kit. This project was a small part of that collaboration. The DNA Identify Laboratory was chosen by Promega to assist with the construction of a population database because of the number of samples available and the need for confirmed father/son pairs. The objectives of the study were to type ~200 father/son pairs from each of the Caucasian and African American races, and then determine the haplotype frequencies, haplotype diversities, and mutation rates for each race.Item Comparison of DNA Extraction Methods From Bone to be Used With the DNA IQ System on the Maxwell 16 for Human Identification(2008-08-01) Lopez, Kristen; John Planz; Arthur Eisenberg; Joseph WarrenLopez, Kristen M., Comparison of DNA Extraction Methods From Bone to be Used with the DNA IQ System on the Maxwell 16 for Human Identification . Masters of Science (Graduate School of Biomedical Sciences), August, 2008, 52 pp., 11 tables, 15 figures, bibliography, 24 titles. Extraction and purification of DNA from human bones is essential for correctly identifying the remains through DNA analysis. Current DNA extraction methods include a demineralization step, which extracts calcium and phosphate from the bone matrix, inactivation of DNAses, and the removal of Polymerase Chain Reaction (PCR) inhibitors. These methods often use harsh chemicals and may allow for residual DNA to be discarded in various wash steps. To assess the effectiveness of DNA extraction from bone samples, two extraction protocols were compared. The first method included a bone demineralization pretreatment solution of Sodium N-Laurylsarcosinate, 0.5 M EDTA, and Proteinase K (20 mg/ml). The second included a pretreatment using a Bone Incubation Buffer by Promega Corporation, with an addition of Proteinase K (18mg/ml). Various incubation times were included to assess the extraction at different time intervals. All extracted samples were purified with the DNA IQ Reference Sample Kit on the automated Maxwell 16 Instrument (Promega Corp.). Full and partial profiles were obtained from samples extracted with the Bone Incubation pretreatment, regardless of incubation time. Profiles were not observed with the standard demineralization pretreatment when amplified at 28 cycles, with partial profiles present in a few samples when amplified at 32 cycles.Item Concordance Study of Forensic Casework Samples Using the AMPFlSTR Kit, AMPFlSTR Identifiler Kit, AMPFlSTR Profiler Plus Kit and PowerPlex 16 Kit(2002-07-01) Armstrong, Treva L.; Arthur Eisenberg; John Planz; Joseph WarrenThe PowerPlex 16, AmpFlSTR Profiler Plus and AmpFlSTR COfiler Kits allow for the co-amplification of the amelogenin gender determining marker and the thirteen core CODIS STR loci: D3S1358, FGA, vWA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, D16S539, THO1, TPOX and CSF1PO. PowerPlex 16 adds the Penta D and Penta E loci and the AmpFlSTR Identifiler addes the D2S1338 and D19S433 loci. Manufactures of these systems have a suggested input DNA sample range of 0.5-2.5 ng, but have been successfully used to type samples containing less than 0.5 ng of DNA. In this study, several questions were addressed: First, “Are all four STR multiple kits concordant in their reproducibility, reliability, sensitivity and efficiency?” Second, “Is one particular STR megaplex kit more applicable to routine forensic casework?” and Third, “In a mixed DNA sample can individuals, whether male or female, be differentiated?” This paper describes a casework concordance study using adjudicated nonprobative sexual assault, mixed DNA and reference blood samples. Amplifications on all samples, were performed using the AmpFlSTR COfiler, Profiler Plus and Identifiler and PowerPlex 16 Kits and genotyping results were obtained using GeneScan and Genotyper software.Item Construction of a Cost Effective Nested-PCR Reaction for Use with the Applied Biosystems AmpFLSTR Identifiler Kit(2005-12-01) Mikeska, Margo M.; John Planz; Joseph Warren; Arthur EisenbergHuman STR analysis has greatly increased the ability to perform identity testing for many different situations. These situations include, but are not limited to, the identification of individuals involved in violent crimes, establishing paternity, and identification of unknown human remains. The most common type of DNA information currently used for identity testing is the short tandem repeat, or STR. STR testing utilizes the number of repeating units in the DNA to assign an allele. Alleles from several different loci are used to establish a genetic profile. Currently, the United States used a standard of 13 different DNA loci to establish identity. These 13 loci can be typed by using a number of different multiplex kits such as the Applied Biosystems Profiler Plus, Cofiler, and Identifiler Kits [1,2]. The 13 loci were selected based on a number of parameters. Each locus was required to be polymorphyic, and a tetranucleotide repeat. The loci also could not display any linkage between each other and extensive population studies had to be conducted to both verify the absence of linkage and to establish allelic frequencies [1]. The goal of this research was the construction of a more cost effective method of utilizing the Applied Biosystems Identifiler Kit. Across the country there is a large backlog of samples that need to be processed in order to obtain a genetic profile. If these samples could be tested using a more cost effective method, more funding could be directed to other endeavors. Paternity testing, as well as some research endeavors could be conducted at a fraction of the cost, leaving resources for other projects or additional staff. Although it would be inadvisable to use this technique on forensic samples, the implications on paternity and research samples would be positive. This research attempted to design a nested PCR reaction and subsequently dilute the Applied Biosystems Primers in order to reduce the cost. The first step was to design new primers for the first round of PCR, followed by testing of those primers. The new primers then required optimization so that they all worked effectively together. After optimization was accomplished, the Identifiler primers were diluted until loci began dropping out of the genetic profile.Item Defining Effective Approaches of Biotech Companies to Commercialize Technology(1998-06-01) Cappelletti, Patricia Margaret Burton; John Licciardone; Robert Gracy; Brenda GriffinCappelletti, Patricia., Defining Effective Approaches of Biotech Companies to Commercialize Technology. Master of Science (Biomedical Sciences), June 1998, 153 pp., 76 tables, reference list, 30 titles. The objective of the study was to define approaches to commercialize technology by biotech companies affiliated with academic institutions, determining the most effective ones. The hypothesis was approaches biotech companies use to commercialize technology their success and a specific combination of approaches would be most successful. A survey provided date from 85 biotech companies on technology origins, patenting, licensing, funding, and product focus. Multiple regression analyses suggested significant relationships between variables and success. Companies with marketed products acquired technology from non-academic sources (p=0.0495), particularly inhouse research/discovery (p=0.0028) rather than other sources. With one academic-sponsored technology, younger companies (≤10 yrs) have a greater probability (74%) of success than older companies (57%). Younger companies with technology transfer offices and companies patenting before publishing are more likely to have marketed products than companies who don’t. Chances of success increase with age and revenues. Results suggest approaches used by biotech companies influence their success.Item Determination of the Intracellular Levels of Cyclic ADP-Ribose in Cultured Human Cells Using a New Highly Sensitive Fluorescent HPLC Method(2002-05-01) Gill, Paramjit Kaur; Goldfarb, Ronald H.; Easom, Richard; Matthew, Porunelloor A.Determination of the Intracellular Levels of Cyclic ADP-Ribose in Cultured Human Cells Using a New Highly Sensitive Fluorescent HPLC Method Paramjit Kaur Gill*. Department of Molecular Biology and Immunology, University of North Texas Health Science Center at Fort Worth, TX 76107-2699. Cyclic ADP-ribose (cADPR) is a second messenger that mobilizes intracellular stores of calcium in higher eukaryotic cells. The intracellular concentration of cADPR has previously been estimated to be in the femto- to nanomolar range. Therefore, there is a need for a highly sensitive assay to measure the levels of nucleotide in just a few million cells. Here, we have developed a highly, sensitive, specific, and reproducible fluorescent HPLC method to determine the intracellular concentration of cADPR in cultured cells. The procedure involves extraction of the nucleotide pool in 20% (W/V) TCA followed by the purification of soluble molecules containing two or more soluble molecules containing two or more riboses by boronate affinity chromatography. Purified nucleotides are then digested with phosphodiesterase to degrade all non-cyclic molecules, leaving cADPR intact. Contaminating products of phosphohydrolysis are then eliminated by a second boronate step and the pure preparation of cADPR obtained is converted to monomeric ADP-ribose with NADase (isolated from Bungarus fasciatus). After a third boronate purification, ADP-ribose is chemically derivitized to the etheno-adenine fluorescent form with chloroacetylaldehyde at 60°C, and the εADP-ribose formed is quantified by fluorescent-HPLC on a Partisil 10-SAX column. The specificity of our method was monitored by determining the yield at every step of the protocol with {32P]cADPR. Radiolabeled cADPR was synthesized from [32P]β-NAD+ and pure ADP-ribosyl cyclase from Aplysia californica. [32P]cADPR was subsequently purified by HPLC on a Partisil 10-SAX and a C-18 reverse phase column placed in tandem. While the recovery of a known amount of cADPR through each boronate step of the 4-day protocol was approximately 90%, the overall recovery throughout the procedure was between 30-40%. As expected, our mock incubations (negative controls) in the absence of phosphodiesterase or NADase treatment, as well as chloroacetaldehyde, yielded no εADP-ribose peak. Furthermore, spiking of a cell extract with commercially available cADPR resulted in the formation of a bigger fluorescent peak. Finally, our method indicated an intracellular concentration of cADPR in HeLa cells of 980 pmol of cADPR/10^8 cells. Considering HeLa cells have a larger cytoplasm compared to blood cells, our results agree well with those reported by DaSilva et al. who observed that the intracellular concentration of cADPR was 198 pmol/10^8 Jurkat cells, using a less sensitive chromatographic assay. Applications of this assay as a tool for biochemical investigation as to the role of cyclic ADP-ribose in the signal transduction events of rapamycin as an immunosuppressant and CD83 in B-CLL are discussed. *The laboratory study in this thesis were performed entirely in the laboratory of Dr. Rafael Alvarez-Gonzalez, in the department of Molecular Biology and Immunology and were performed entirely under his supervision. Dr. Alvarez served as my mentor from 1995-1998. Both Drs. Rafael Alvarez-Gonzalez and Ronald Goldfarb, Department Chairman, served as co-mentors from August 1999-March of 2000. From April 2001-July 2001, Dr. Goldfarb served as my mentor.Item Evaluation and Validation of Tecan Genios Microplate Reader for Quantification and Normalization of Family Reference DNA Samples(2007-08-01) Fuqua, Lauren; John Planz; Arthur Eisenberg; Joseph WarrenIn 2001, the Texas State legislation established the Texas Missing Persons DNA Database (TMPDD) at the University of North Texas System Center for Human Identification Laboratory. Texas was the first state to participate in the missing persons section of the federal (FBI) database titles Combined DNA Index System or CODIS. Two indices of CODIS include the Unidentified Human Remains index and the Relatives of Missing Person index. Medical specimens, such as bone marrow or blood, or personal items used only by the missing person, such as a toothbrush or hairbrush, are ideal for identifying human remains through comparison of DNA profiles; although, DNA samples can be taken from family members to help locate missing persons or identify remains. DNA profiles from family reference samples, such as blood or buccal swabs from a close relative, are analyzed and uploaded into CODIS to allow federal, state, and local crime laboratories to exchange and compare profiles to missing persons electronically. At the University of North Texas Health Science Center, family reference samples, missing person reference samples, and unidentified human remains are analyzed to obtain DNA profiles for comparison. This research project involves a method that is proposed to improve the efficiency of DNA analysis for family reference samples. At the UNT System Center for Human Identification laboratory, the family reference samples are extracted in batches of 86 using the Tecan Freedom EVO® 100 extraction robot with the DNA IQ™ extraction kit from Promega Corporation. The DNA IQ™ extraction process is used in conjunction with the EVO® 100 robot in order to obtain a consistent amount of total extracted DNA; although, substantial variation has been detected in the output DNA quantity delivered. A considerable percentage (~20%) of samples exceed the optimal input template DNA amount required for successful amplification using the Applied Biosystems AmpFʅSTR® kits. A method of normalizing these samples was needed to bring the standard input DNA range within the optimal analytical range of the Applied Biosystems 3130 Genetic Analyzers and GeneMapper™ ID software. The ultimate objective of this internship practicum was to improve the efficiency of DNA analysis for family reference samples by using the Tecan GENios microplate reader in conjunction with an OliGreen® assay to estimate DNA quantity with the aim of using the quantification values to normalize family reference samples into an ideal input range for genetic analysis.Item Evaluation of a Novel Dual Biometric Device Used to Obtain DNA from Fingerprints(2008-06-01) Mann, Michael L.; Arthur Eisenberg; John Planz; Joseph WarrenSummary: MICROFIELD COMPANY S.A. is currently developing a non intrusive method of obtaining a reference DNA sample known as Lift & Rub (Figure 1). The Lift & Rub is a self-adhesive security stamp designed to collect a single fingerprint from a known individual that can be used for DNA analyses. Lift & Rub is composed of an adhesive material with a protective film. The Lift & Rub is composed of an adhesive material with a protective film. The Lift & Rub is used in conjunction with an abrasive strip and a product named Dry-Ink, a graphite laminate. Together these items allow for an alternative to the traditional fingerprint collection technique and improve on it by allowing for DNA analysis. The objective of this research is test and maximize the performance of Lift & Rub. Microfield’s Lift & Rub is designed to collect a single fingerprint from a known individual to be used for DNA analyses. The Lift & Rub is intended to provide a full genetic profile of a known individual. This project aims to: 1) establish a standard collection protocol by evaluating empirical data to determine the average DNA yields using various collection techniques; 2) evaluate the reproducibility of DNA profiles obtained; and 3) gauge the overall success rate of obtaining a complete profile.Item Evaluation of a Novel Multiplex Ministr System for Analysis of Degraded and Low Copy DNA Samples(2006-05-01) Orcutt, Joseph L.; Arthur Eisenberg; Joseph Warren; John PlanzOrcutt, Joseph L., Evaluation of a Novel Multiplex MiniSTR System for Analysis of Degraded and Low Copy DNA Samples. Masters of Science (Forensic Genetics), May, 2006, 78 pp., 25 tables, 14 figures, bibliography, 20 titles. The goal was to evaluate the performance of a novel miniSTR multiplex system for the analysis of degraded and low quantity DNA samples. Three studies were designed to evaluate this new miniSTR kit: 1. A concordance study to insure that the profiles generated are identical to those with currently used STR kits; 2. A dilution study to identify the sensitivity limits of the multiplex system, and 3. The ability to generate profiles from DNA isolated from skeletal remains which had previously given incomplete profiles using conventional STR kits. The results indicate that the Applied Biosystems new miniSTR multiplex system will provide a valuable tool for forensic scientists to obtain genetic data from challenging casework samples.Item Evaluation of Applied Biosystems' Real-Time Human Quantification Assays(2003-05-01) Hybki, Dixie Lee Peters; John Planz; Arthur Eisenberg; Joseph WarrenTo aid the forensic community with its quantification issues, Applied Biosystems is currently developing human specific and human male specific quantification assays using Real-Time PCR (RT-PCR) and TaqMan probes. The human specific assay amplifies an autosomal specific gene, located on chromosome five, while the human male specific assay amplifies a region on the Y chromosome. The purpose of this project was to evaluate the assays with forensic samples to determine if the use of these kits would be appropriate to the forensic community. These kits are not commercially available at the time of this writing. Therefore, several details have been omitted to protect the patent and legal issues that are still pending. It is expected that these assays will surpass the sensitivity and specificity of current methods. This will not only meet, but also exceed the standard set forth by the DAB. By providing additional information such as human male DNA quantification and PCR inhibitor detection, these kits can provide what the forensic community has been lacking. The human male DNA detection and quantification is valuable in providing proof that male DNA was present in an intimate sample from a sexual assault case. This would be especially important in a case in which the offender was a vasectomized male, and for resolving mixtures of the victim and offender’s genetic profiles. The detection of PCR inhibitors for the elimination of futile genetic analysis is a novel component that would provide additional advantages. These kits will offer means for proper quantification to allow for minimal space waste, and allow for successful multiplex PCR within its optimal range. Today, STR analysis will proceed, and is often successful, even if no quantification results are obtained with current methods. The legal system questions this approach. The ability of autosomal specific and Y-chromosome specific RT-PCR quantification assays to assess low level DNA would provide the justification for subsequent analysis that would quiet the legal system’s arguments concerning human quantification.Item Evaluation of Genomiphi Whole Genome Amplification Kit for Use in Low Copy Number Forensic Cases(2004-12-01) Halsell, Lloyd F.; John Planz; Joseph Warren; Arthur EisenbergAmersham Bioscience produces the only available WGA kit, GenomiPhi DNA Amplification kit. It is a multiple displacement amplification reaction. The kit contains all reagents needed to perform WGA. Optimal amount of DNA input into the reaction is 1 ng or greater to yield 3 μg product. This amount is the optimal amount currently needed by PCR systems; however STR amplification requires the 1 ng in 10 μL of sample extract. GenomiPhi requires 1 μL of sample extract, ten times the concentrations of STR systems. The goal of this project was to evaluate the possible uses of the GenomiPhi DNA Amplification kit for use in the forensic community. The ability to amplify the entire genome without bias would benefit low copy number samples where there is little DNA to start with. One objective of the study was to determine the lower limit of input DNA into the GenomiPhi reaction. Input DNA varied from 10 ng to 7 pg. Secondly, input DNA was degraded to determine how the GenomiPhi kit will be affected by the input of less than pristine DNA. All samples were quantitated by PicoGreen assay, STR amplified with Profiler Plus and analyzed on ABI’s 310 Genetic Analyzer. Samples were analyzed before and after WGA to determine under which circumstance better results were seen.Item Evaluation of SPEX6750 Freezer Mill Pulveration of Teeth for Use in Nuclear Casework at the Armed Forces DNA Identification Laboratory(2008-08-01) Bintrim, Kristin A.; John Planz; Arthur Eisenberg; Joseph WarrenBintrim, Kristin A., Validation of Freezer Mill Pulveration of Teeth for use in Nuclear Casework at the Armed Forces DNA Identification Laboratory. Master of Science (Forensic Genetics), August, 2008, 44 pp., 4 tables, 6 figures, bibliography, 27 titles. I completed a validation study at the Armed Forces DNA Identification Laboratory. It was an evaluation of freezer mill pulveration of teeth for use in nuclear casework. The teeth were cleaned using a bleach and sonication method and then ground using a freezer mill. DNA extraction was achieved by using silica based capture techniques. The DNA was quantified, amplified and evaluated using fragment analysis. Several different amounts of input tooth powder were attempted to increase the DNA yield. Qiagen MinElute produced higher DNA yields then Promega’s DNA IQ system for the small sample size tested. After reviewing the results it was concluded that the DNA was most likely highly degraded and therefore did not produce complete genetic profiles for the samples tested.Item Evaluation of the AluQuant Human DNA Quantitation System Using the 96-Well Plate Format(2002-08-01) Alexander, Uvonna Faye Lewallen; John Planz; Arthur EisenbergAlexander, Uvonna Faye Lewallen, Evaluation of the AluQuant Human DNA Quantitation System Using the 96-Well Plate Format. Master of Science (Forensic Genetics), August 2002, 121 pp., 4 charts, 4 tables, 8 figures, 2 appendices, references, 13 titles. This study evaluated the AluQuant Human DNA Quantitation System (Promega Corporation, Madison, WI) using the 96-well plate format for possible implementation by Orchid Cellmark Dallas. The importance of human DNA quantitation in forensics is two-fold. First, the Quality Assurance Standards set forth by the DNA Advisory Board requires human DNA in forensic samples be quantitated. Also, the highly sensitive PCR multiplex PCR multiplex assays used in forensics have been optimized for a narrow range of template DNA, thus requiring accurate and consistent quantitation. This evaluation consisted of three general goals: examination of the Reporter Microplate Luminometer (Turner BioSystems, Sunnyvale, CA), alteration of the assay variables to obtain optimal performance, and characterization of the assay. The Reporter produces reproducible results and is sensitive to at least 4.88 x 10^-9 moles ATP. Of the variables tested, quick centrifugation of the incubation plate had the most noticeable effect on the results obtained. The assay did not perform as characterized by Promega. AluQuant is not reproducible, nor does it consistently produce results within a two-fold accuracy range. Therefore, Orchid Cellmark Dallas will not be implementing the AluQuant Assay.Item Evaluation of the SlicPrep 96 Device for Use in Extraction of Forensic Samples on the Biomek 2000 Laboratory Automation Workstation(2005-07-01) Plopper, Farah Jo Homsi; Joseph Warren; Arthur Eisenberg; John PlanzThe purpose of this study was to evaluate the Slicprep ™ 96 Device for extraction of forensic samples on the BioMek® 2000 robot to determine if it can further automate and increase the efficiency of the extraction process as well as generate optimal DNA yields with no contamination. This validation study was not as comprehensive as PBSO's validation of the BioMek® 2000, because the platform and reagents used were the same. The differences include the use of the Slicprep™ 96 Device during extraction and the slight modifications to protocol that were made to accommodate this device.Item Evaluation of Unlabeled Primers for Sex Determination of Animal and Wildlife Samples(2006-07-01) Price, Glynis Nicola; John Planz; Arthur Eisenberg; Joseph WarrenSpecies identification is not only important in determining the origins of remains found in human forensic cases, but is also important in the growing field of nonhuman forensics. Animal forensics is an emerging discipline in the non-human forensics field. Animal forensics focuses on domestic animals, or animals that live in close contact with humans. These animals include dogs, cats, cattle, pigs, horses, sheep as well as others. Objective 1: Sensitivity - Forensic samples are often low copy number so there may be little viable or non-degraded DNA to work with. The PCR reaction needs to be optimized to determine how little DNA you can start with and still obtain accurate results. The given protocol from DNA Solutions, Inc. (Appendix A) [ 17] suggests 20ng of input DNA. In practice, this level is rarely found in forensic cases, so this assay will begin at 5ng of 9 input DNA. The dilution series will be 5ng, 2ng, 1 ng, 500pg, 250pg, 125pg, 62.5pg, 31.25pg and 15.63pg. Objective 2:PCR cycle number- As a part of sensitivity, PCR cycle number is an important part of optimizing the reaction. Since agarose gels are being run to visualize results, increased cycle number can result in increased specificity. Cycle numbers being evaluated are 26 cycles [ 17], 28 cycles, 30 cycles, 32 cycles and 34 cycles. Objective 3: Species Specificity - It must be determined if these universal primers do, in fact, bind to all species that possess the genes of interest. Samples from across the different animal classes will be evaluated to determine the specificity of the primers. The primers have already been tested on members of the deer family, with success, so a male deer sample has been sent to be used as a positive control as it shows both genes. Species of interest are mammals, birds and fish as there are many of each that are endangered and identifying the sex of the animal is an important step to identifying the individual animal.
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