Browsing by Subject "Forensic Science and Technology"
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Item A DNA-Based Multiplex Screening Tool for Separation of Fragmented and Commingled Skeletal Remains(2007-12-01) Ambers, Angie; Joseph Warren; John Planz; Arthur EisenbergAmbers, Angie, A DNA-based Multiplex Screening Tool for Separation of Fragmented and Commingled Skeletal Remains. Master of Science (Forensic Genetics), December, 2007, 63 pages, 13 tables, 19 figures, references, 38 titles. In mass death scenarios, human remains are often fragmented, scattered, and commingled. Ascertaining the number of victims and determining the victims’ identities in such scenarios is a challenging task. A DNA-based screening tool used early in the investigation of mass disasters or mass graves would provide a relatively quick way to initially assess casualty numbers and separate remains for further analysis. Such a tool would promote the most efficient allocation of resources and speed the identification process. The multiplex designed here incorporates a few genetic loci that show high variability in the human population, giving it sufficient discriminatory power for separation of commingled remains. Specifically, the multiplex includes the amelogenin sex-determining locus, D3S1358, and a 3’ (CA)n dinucleotide repeat in the mitochondrial D-loop. Further optimization/validation studies need to be conducted, and a fourth locus (D5S818) may need to be considered to increase the tool’s power of discrimination.Item Amplification of Mitochondrial DNA Regions HVI and HVII in its Entirety and Reducing Cycle Sequencing(2004-08-01) Ariyo, Bolanle; Joseph Warren; John Planz; Arthur EisenbergAriyo, Bolanle. Amplification of Mitochondrial DNA Regions HVI and HVII in its Entirety and Reducing Cycle Sequencing Reactions. Master of Science (Forensic Genetics), August 2004, 46 pages, 10 figures, 7 tables, 18 references. Mitochondrial DNA is widely used in the forensic community because of its high copy number in cells, location, and mode of inheritance. Yet this method of analysis is expensive, time consuming, and labor intensive, therefore labs should take steps to improve the procedure of mtDNA analysis. This study is performed to validate the use of amplifying HVI and HVII region in its entirety (2 primer sets) for use in reference samples. Amplification performed using primers F15989-R16410 (HVI) and F73-R340 (HVII). The current method of amplification is 4 primer sets at full cycle sequencing reactions. The cost of Cycle Sequencing Kit is also expensive, therefore performing half and quarter reactions would be beneficial in reducing the amount of kit consumed. To validate the use of reducing cycle sequencing reactions, half and quarter cycle reactions were performed using 2 and 4 primer sets. Results demonstrate that sequence data for reducing cycle sequence data is consistent with the sequence data using the current method. Results also show that sequence data obtained using two primer sets was consistent with sequence data amplified by the current method with the exception of two samples at length heteroplasmy polyctosine regions.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 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 Cell line authentication and contamination assessment for human cell cultures(2015-05-01) Ormos, Andrea; Arthur J. Eisenberg; Rhonda Roby; John V. PlanzCell line authentication is an essential step in ensuring the integrity and reproducibility of biomedical research. The major contaminants in cell cultures are fungi, viruses, bacteria and contamination from other cell lines of the same or different species. Contaminants alter the physiology and properties of cells, compromising the results of experiments. In this study, an improved multiplex assay was developed, detecting mycoplasma and mouse cell line contamination, while performing DNA typing. The assay was tested on cell cultures, the reproducibility of the assay was verified, sample collection and procedures were optimized and limit of detection for contaminants were determined. A survey was conducted to assess the interest in an in-house cell line authentication and contamination assessment service.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 Comparison of Next Generation Sequencing Methodology on the Ion PGM™ System Performance versus that on the Sanger Sequencing Method for HV1 and HV2 Regions of mtDNA(2015-05-01) Argueta, Wendy C.; Arthur J. Eisenberg; Michael Allen; Raghu R. KrishnamoorthyAnalysis of mitochondrial DNA in forensic applications has enabled the identification of a missing person through comparison with additional maternal relatives. Most forensic applications are based on sequencing of both hypervariable regions of the mtDNA. Sequencing of these regions has been commonly done using Sanger-type sequencing (STS) methodology, which is expensive, time-consuming and laborious. Next Generation Sequencing (NGS) technology, such as the Ion Torrent PGM™ System platform, overcomes most of these issues. In this study, samples from the Guatemalan population (n=40) were sequenced with both Ion Torrent PGM™ technology and STS methods. A high level of consistency (98%) was observed among data derived from both methods. Most of the discrepancies were point heteroplasmy, which were more easily detected by PGM™ technology. In terms of performance, the NGS method was shown to be quick, with high-throughput and more efficient compared to the traditional STS method. More accurate and reliable sequencing data were obtained from the Ion Torrent PGM™ method due to its high level of coverage. Sequencing data for all individuals, representing 19 different family groups, were obtained using the NGS technology. Sequence polymorphisms were detected in 55 positions, from which 26 were observed only in relatives belonging to the same family and were not observed for any other family group. In a forensic context, haplotype specific polymorphisms are the basis for identification and comparison between evidence and reference samples purposes. Haplotypes between maternally related individuals were consistent in 18 family groups.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 Development of a Comprehensive Massively Parallel Sequencing Panel of Single Nucleotide Polymorphism and Short Tandem Repeat Markers for Human Identification(2015-08-01) Warshauer, David H.; Bruce Budowle; Ranajit Chakraborty; Bobby L. LaRueMassively parallel sequencing (MPS) technologies allow for the detection of an unparalleled amount of genetic information with unprecedented speed and relative ease. These qualities make the technology desirable for generating DNA profiles that may be uploaded into forensic offender, arrestee, and family reference database files. This doctoral dissertation research was conducted under the hypothesis that MPS, with its exquisitely high throughput, can provide a system whereby reference samples can be typed for a large battery of markers, providing more discrimination power for forensic DNA typing and offering increased opportunities to develop investigative leads. The design and implementation of large marker panels for the typing of reference samples will reduce debates on the best core markers for forensic utility, generate innovation because focus will not be solely on a core set of autosomal STRs, promote the development of better systems that can analyze more challenging samples, and enable sharing of data across laboratories worldwide. The primary goal of this project was to develop the capability of typing reference samples for a large battery of markers: 84 autosomal, Y-chromosome, and X-chromosome short tandem repeats (STRs), Amelogenin, and 275 human identity single nucleotide polymorphisms (SNPs), in a single multiplex analysis. To that end, a bioinformatic software package, STRait Razor, was developed to detect STR alleles in raw MPS data. A proof-of-concept study was performed to evaluate the efficacy of using MPS to type forensically relevant markers, using a PCR multiplex-based SNP assay. The proposed comprehensive capture-based MPS panel then was designed and extensively tested. Finally, the benefits of the additional genetic data afforded by MPS, as opposed to traditional methods, were illustrated through the characterization of intra-repeat nucleotide variation within Y-chromosome STR alleles. The results of this dissertation research indicate that MPS is capable of providing robust genetic data from a wide variety of forensically-relevant STR and SNP loci in a single analysis. To date, the comprehensive MPS panel developed during the course of these studies is the most potentially informative assay for reference sample testing for human identification.Item Developmental and Internal Validation of a Mitochondrial DNA Direct Amplification Kit for Forensic Reference Samples(2015-05-01) Alicea-Centeno, Alessandra; Arthur J. Eisenberg; Rhonda Roby; Dixie L. PetersEvaluation of the control region of the mitochondrial genome is a common practice for forensic casework and research purposes. Since no kit is currently commercially available for the amplification of mitochondrial DNA (mtDNA), its sequencing procedure is time-consuming and laborious. Six steps are generally followed: DNA extraction, quantification and normalization, amplification of two regions (hypervariable regions 1 and 2), cycle sequencing, capillary electrophoresis and data analysis. This project evaluated a mtDNA direct amplification kit by performing developmental and internal validations. The studies performed included sensitivity, stability, reproducibility, case- type samples, mixtures and accuracy. The mtDNA direct amplification kit successfully amplified reference samples used in each study without the need of extraction and quantification steps. In addition, mtDNA profiles were obtained from the sequenced amplification products. Using the validated direct amplification procedure in the laboratory will improve workflow, decrease operational cost and reduce the possibility of error by minimizing sample handling.Item Discovery and Characterization of Tetranucleotide Short Tandem Repeats in North American Bears (Ursids)(2015-05-01) Graham, Michelle L.; John V. PlanzAccurate individual identification is essential to wildlife crime investigations and conservation genetics. Current methodology utilizes dinucleotide short tandem repeats (STRs) that can be difficult to type accurately and have high mutation rates; however, tetranucleotide STRs have greater stability and allele diversity. The main objective of this study was to identify potential tetranucleotide STR loci and internal variants for the American black bear, brown bear, and polar bear. Barcoded genome libraries were prepared for each species from extracted and enzymatically fragmented DNA, size selected, quantified, enriched using biotinylated RNA baits to capture twelve common mammalian sequence motifs, and massively parallel sequenced. One potential locus was identified using the NextGENe® software and six potential loci were identified using algorithm mining.Item Effect of Collagenase Type 2 and Proteinase K Digestion on DNA Yield from Bone Samples Purified on the EZ1 Advanced XL(2015-08-01) Barrett, Lisa C.; Arthur J. Eisenberg; Joseph E. Warren; Raghu R. KrishnamoorthyGenetic results from bone samples often yield low quantities of DNA and poor quality of genetic data. Proteinase K is a proteinase that is commonly used in DNA extraction methods, however the target proteins of Proteinase K do not closely align with the makeup of bone. Collagenase Type 2 is a protease that is more specific to the breakdown of collagen, which bone is comprised of. This study looked at the potential effects of Collagenase Type 2 digestion on bone samples compared to the effects of Proteinase K on quantity and quality of genetic typing. This study also incorporates the EZ1 Advanced XL purification platform and the AmpFLSTR Globalfiler Amplification Kit.Item Evaluating Noise and the Implications of Methodology in the Analytical Threshold Designation for Forensic Genetic Analysis(2015-08-01) Malone, Ashley N.; Joseph E. Warren; John V. Planz; Raghu R. KrishnamoorthyIt is common in the forensic science community to have standardization and uniformity in all laboratory processes. The method for the determination of a minimum detection threshold or synonymously an analytical threshold for genetic analysis is not uniform across forensic labs. Variation amongst the methods in DNA testing by forensic laboratories leads to variations in the results of the DNA testing. The results of this study show a method using DNA sample types versus non-DNA sample types will better reflect the effects of baseline noise that may be encountered in forensic casework samples. In addition, there is a need for a calculation method to be designated as an appropriate tool in determining analytical thresholds. More studies on baseline noise and methods in distinguishing analytical thresholds will help in the determination of the most appropriate calculation method to be used across all forensic laboratories.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 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 Kinship Indices for the Identification of Missing Persons(2006-07-01) Hamilton, Kristi Payne; John Planz; Arthur Eisenberg; Joseph WarrenWhen both parents are able to provide reference DNA samples, the likelihood ratio, or strength of a match, between the parents and their child will be very high for a true match. However, what happens when only one parent is available, or neither parent is available? How effective are other relatives’ DNA profiles at aiding in the identification of an unknown sample? What sort of threshold, if any, should be in place to determine whether an unknown is excluded from being the relative of the reference donor? This study aims to approach an answer these questions by analyzing a database consisted of anonymous samples from the paternity testing division of the University of North Texas Health Science Center DNA Identity Laboratory. The concentration of the study is on whether kinship indices are reliable and consistent in being able to provide information regarding a sibling relationship. Evaluation of the kinship indices of parents and siblings of different families, both within the family and outside of the family, will aid in the determination of whether or not a threshold exists, and what that threshold may be. This information will be invaluable to future cases involving unidentified remains when direct reference samples or parent reference samples are not available.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.
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