Browsing by Subject "DNA"
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Item A Comparative Study of Three Methods to Enhance the Collection of DNA from Plant Material(2013-05-01) Ausmer, Alea D.; Warren, JosephThe Botanical Research Institute of Texas is using two methods of DNA extraction from plants, an automated method called Bullet Blender® and a manual method of grinding. A third method, using an instrument called the Fast Prep-24™, was evaluated and the DNA yield obtained was compared to the other methods. Eight plant species were chosen and two sample preparation methods, wet and dry, were evaluated. DNA yield gels were run in order to compare DNA quality and UV spectroscopy was used to evaluate quantity. Independent Student t-tests were performed to compare means variation between the DNA yield on the wet and dry samples and one-way ANOVA was used to compare variation between the three extraction methods. No significant difference was found between the wet and dry samples for DNA concentrations, but a significant difference was observed between the Fast Prep-24™ instrument and the other two methods.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 A Semi-Automated Methodology for Extraction of DNA from Human Skeletal Remains(2013-05-01) Mize, Mary L.; Arthur EisenbergThe current methodology used by the University of North Texas Center for Human Identification Missing Persons Laboratory (UNTCHI) to recover DNA from skeletal remains is time-consuming, laborious and not readily amenable to automation. The constraints of the current process limit the number of samples that can be analyzed. The results of this study show that extractions performed with the AutoMate Express™ Forensic DNA Extraction System (Life Technologies, Carlsbad, CA) can produce comparable DNA quantity and quality to the current procedure used by UNTCHI. The utilization of the AutoMate Express™ Forensic DNA Extraction System in our operational laboratories would help streamline the process of DNA extraction from human skeletal remains and potentially provide increased amounts of genetic information.Item A SEMI-AUTOMATED METHODOLOGY FOR THE EXTRACTION OF DNA FROM HUMAN SKELETAL REMAINS(2013-04-12) Mize, MaryPurpose: The conventional method used to extract DNA from skeletal remains is not readily amenable to automation. The numerous steps in the current procedure increase the risk of both sample loss and the potential for contamination with extraneous DNA. An automated DNA extraction procedure will be evaluated and optimized for the quantity and quality of DNA recovered from bone samples on the AutoMate Express™ (Life Technologies). The goal of this project is to determine if this process will yield equivalent amounts of DNA that will produce autosomal STR profiles and mitochondrial DNA sequence data that are comparable to those obtained with the conventional method of organic extraction. Methods: Varying amounts of bone powder, from different age groups of remains, were incubated with BTA Lysis Buffer (Life Technologies) which contains DTT and Proteinase K from two hours to overnight. After the incubation any remaining bone powder was spun down and 230µL of the lysate was loaded onto the AutoMate Express™ columns containing the PrepFiler™ magnetic beads. Total run-time for the instrument is 30 minutes and DNA is eluted in 50µL of buffer. The extracts were quantified and amplifications were performed. Autosomal STR profiles and mitochondrial DNA sequence data generated from samples processed using the AutoMate Express™ were compared to the data generated by the conventional organic extraction method. Results: No significant difference was observed between a 2-hour incubation and overnight incubation when using the AutoMate Express. The overall quantity and quality of the DNA extracted using the AutoMate Express™ was equal to or better than the DNA extracted using the conventional organic method. Conclusions: To this point the DNA obtained using the AutoMate Express, which takes a total of 4 hours, generates genetic data that is comparable or better than the data generated using the conventional methodology which takes 2-3 days.Item A Validation of STRmix™ for Forensic Casework(2017-05-01) Conway, Allison; Bruce Budowle; Joseph E. Warren; Patricia A. GwirtzThe interpretation of complex DNA profiles has been a controversial issue in forensic biology. The current methods of interpretation are limited in scope and do not make full use of the data. Efforts have been made to develop software which can incorporate more data and process more calculations than were previously possible. One such program, STRmix™ (Institute of Environmental Science and Research, Porirua, New Zealand), uses a Markov Chain Monte Carlo (MCMC) principle to simulate many different possible profiles and determine the probability of observing a profile in the evidence. A validation of this software is necessary to indicate which situations are appropriate for analysis and define existing limitations.Item Analysis of a Tn917 Transposon Mutant and Preliminary Characterization of NonHemolytic, Catalase-Deficient Variants of Staphylococcus aureus(1999-06-01) Crum, Russell M.Crum, Russell M., Analysis of a Tn917 Transposon Mutant and Preliminary Characterization of Nonhemolytic, Catalase-Deficient Variants of Staphylococcus aureus. Masters of Science (Microbiology). June 1999. Pages-101. Tables-15. Figures-10. A Tn917 transposon mutant of Staphylococcus aureus S6C was isolated and analyzed due to its deficiency in hemolysin and lipase activities. The transposon insertion did not occur in any of the known genetic regulators, which suggested the insertion occurred in a novel regulator of at least, hemolysin and lipase activities. One end of the region where the insertion occurred was isolated, sequenced, and compared with known DNA databases. Sequence comparisons revealed the insertion occurred in one of six rRNA DNA operons, which was confirmed by Southern analysis. Transduction of the transposon insertion back into the parental strain did not result in a mutant phenotype thereby indicating that the transposon insertion into a rRNA DNA operon was not responsible for the observed mutant phenotype. Further analysis of the parent strain, S. aureus S6C, revealed a population of four relatively stable variants differing in their hemolysin and catalase activities. These data suggest that the Tn917 mutant was one of these four S6C variants.Item Analysis of HemaSpot[TM]-HF and HemaSpot[TM]-HD Sampling Kits Using Trace DNA(2019-05) McGehee, Rachel L.; Warren, Joseph E.; Planz, John V.; Schiro, GeorgeSpot On Sciences, Inc. has recently developed a new device, the HemaSpot[TM], which allows for samples to be stored safely at ambient temperatures. The UNTHSC FGEN program was contacted to conduct a research study to determine its feasibility for use as collection and storage media with trace samples. Extractions of 108 samples were conducted with QIAGEN[R] QIAamp DNA Investigator Kits, a 3130xL Genetic Analyzer, and GeneMapper[R] ID-X software. A hypersensitivity study worked with sub-optimal amounts of control DNA in order to observe the quality and variation of the generated profiles. The trace study swabbed items found at typical crime scenes and determined the device's ability to generate readable profiles. Results uncovered that all samples either contained large portions of allelic dropout or contamination. Relatively similar partial profiles were produced for both cartridge types in the hypersensitivity study. In addition, readable trace profiles were compared to one another to conclude that the HemaSpot[TM]-HD had the most success, however this may have been the cause of limited size and sample variation. Both products should be tested further.Item Assessment of DNA transfer Involving Routine Human Behavior(2010-05-01) Kelley, Shamika; Bruce BudowleDNA transfer events are affected by routine human actions and can impact the interpretation of forensic evidence results. Some scientists have inferred that secondary transfer events lead to only minimal amount of DNA yield and when two people are involved, the DNA profile of the primary person who had contact with the item is typically prominent. To assess the effects of secondary DNA transfer events on DNA quantity, methods similar to those of Lowe et al. [1] were used. We have recruited 12 volunteers (subjects) to participate in a 4-part study consisting of every day human routine behaviors. These routine behaviors include handshaking, holding a pen in the mouth, and licking the thumb before turning the page. Sufficient quantities of DNA were obtained via secondary and tertiary transfer. DNA profiles could be observed from an individual to an object even though that individual did not directly touch the object.Item Automodification Reaction of PARP-1 Reversibly Regulates the DNA-Binding of NF-kB(2001-11-01) Chang, Woo-Jin; Alvarez, Rafael; Mathew, Porunelloor A.; Goldfarb, Ronald H.Chang, Woo-Jin, Automodification Reaction of PARP-1 Reversibly Regulates the DNA-Binding of NF-kB, Doctor of Philosophy (Microbiology and Immunology), November, 2001, 92 Pages, 20 figures, 3 schemes, and bibliography. Poly(ADP-ribose) polymerase (PARP-1, E.C. 2.4.2.30) is a constitutively expressed nuclear enzyme. It comprises about 1% of the total nuclear protein and in phylogenetically well conserved in most eukaryotes, with a notable exception in yeast. PARP-1 post transitionally modifies DNA-binding proteins by transferring the ADP-ribose moiety from BNAD+. Although the exact biological function of poly(ADP-ribosyl)ation has not been clearly elucidated, the process is thought to be involved in DNA repair, replication, and gene expression. Previous studies have indicated that PARP-1 participates in eukaryotic gene expression including the genes under the control of nuclear factor-kB (NF-kB). It has been demonstrated that PARP-1 deficient mice are more resistant to lipopolysaccharide-induced endotoxic shock than isogenic wild-type mice due to the inactivation of NP-kB in the mutants. In order to further analyze the interactions between PARP-1, NF-kB, and its consensus DNA in a cell-free system, we co-incubated recombinant PARP-1 protein and the p50-subunit of NF-kB (NF-kB-p50) in the absence of DNA strand-breaks. Electrophoretic mobility shift assays (EMSA) showed that sequence-specific DNA-binding of NF-kB-p50 was dependent on autopoly(ADP-ribosyl)ation of PARP-1. The NF-kB-p50 DNA-binding was inhibitied when PARP-1 was not auto-poly(ADP-ribosyl)ated either in the absence of BNAD+ or in the presence of 3-aminobenzamide, an enzymatic inhibitor of PARP-1. Coimmunoprecipation and immunoblot analysis demonstrated that NF-kB-p50 formed a heterodimer with PARP-1 when PARP-1 was not auto-poly(ADP-ribosyl)ated. In addition, poly(ADP-ribosyl)ation assays showed that NF-kB-p50 protein was not susceptible to poly(ADP-ribosyl)ation under normal incubation conditions. Those in vitro observations described above were confirmed by experiments utilizing HeLa nuclear extracts. EMSA showed that NF-kB DNA-binding was inhibited in 3-AB-pre-treated HeLa cells. To our knowledge, this is the first report demonstrating that auto-poly(ADP-ribosyl)ation reaction by PARP-1 reversibly regulates the function of a transcription factor by inhibiting the formation of heterodimer between PARP-1 and a transcription factor.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 Four Differential DNA Extraction Methods for Casework Analysis of Sexual Assault Kit Swabs(2016-05-01) Brignac, Francine J.; Joseph E. WarrenSexual assault kits make up 40-50% of a typical Forensic Laboratory caseload. The traditional method to process these samples is time-consuming and requires the use of hazardous chemicals such as Phenol:Chloroform:Isoamyl Alcohol (PCIA). This study compares another manual differential extraction method and two automated methods to the traditional standard differential extraction. Results indicate that as sperm sample concentration decreases, automated methods produce superior results both in DNA quantity obtained and in quality of STR profiles produced. Automated methods reduce hands-on time, facilitate higher through-put of samples, and reduce analyst contact with hazardous chemicals such as PCIA, making it an excellent choice for labs.Item Compiling an Autosomal and Y Chromosomal DNA Database for the Country of Sri Lanka.(2009-08-01) Cherian, Holly; Warren, JosephThe purpose of this project was to compile an autosomal STR and Y chromosomal STR DNA database for Sri Lanka. Profiles from previous processing that are not interpretable will be redone. Additionally, on 10% of the samples a concordance study was conducted to check for reliability. The results proved to be concordant. Testing for Hardy Weinberg Equilibrium was conducted on the autosomal data. The database demonstrated to be in Hardy Weinberg Equilibrium, no linkage disequilibrium was observed, and there was no evidence of inbreeding present. A report was generated including tables of autosomal STR allele frequencies and summary of Y-STR haplotypes. Y-chromosomal haplotypes were reported and the population shows high genetic diversity with 168 haplotypes present.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 Detecting and Quantifying Oxidative DNA Damage using MinION Nanopore Sequencing(2018-05) Blessing, Alexandra M.; Phillips, Nicole; Planz, John; Allen, Michael; He, ShaoqingA common biomarker of damaged DNA, particularly mitochondrial DNA, 8-oxoguanine (8-oxoG) has been identified as a possible contributor to neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, preeclampsia, as well as type 1 and type 2 diabetes. Numerous methods have been developed to detect oxidative damage within the genome, including but not limited to immunological techniques, quantitative-polymerase chain reaction (qPCR), and in situ imaging. This study explores nanopore sequencing using the MinION Nanopore (Oxford Nanopore Technologies, Oxford, UK) as a more sensitive method of 8-oxoguanine detection, providing proof-of-concept for model training as well as preliminary model development.Item Effect of Detergent Selection on Quantity of DNA Obtained and on STR Profile Developed from Bone-Derived DNA(2016-05-01) Proctor, Frances N.; Joseph E. Warren; Bobby L. LaRue; Raghu R. KrishnamoorthyBone is sometimes the only source of DNA in cases of unidentified persons, missing persons, and mass fatality incidents, but bone characteristically provides lower quantities of DNA and lower quality short tandem repeat (STR) profiles than that of other sample types. Bone composition is very different from that of blood and soft tissue, therefore using a method designed for these other sample types is less effective for bone. A bone-specific extraction method is needed in order to improve these results. This study investigated the effects of detergent selection on both the quantity of DNA obtained and on the STR profiles produced from bone-derived DNA, as a part of developing a bone-specific DNA extraction protocol.Item Evaluation of the AmpFlSTR® MiniFiler Typing Kit: Mixture Studies and Non-Probative Sample Studies(2009-05-01) Feller, Elizabeth A.; Planz, John V.This project was aimed to assemble and assess data from internal validations of AmpFlSTR® MiniFilerTM PCR Amplification Kit by forensic laboratories across the United States. After compilation, data was evaluated for quality of testing, results, and concordance within and between participating laboratories. It was concluded that MiniFilerTM can successfully amplify DNA from multiple sources in mixtures of neat and degraded samples, as well as enhance DNA profiles obtained for several types of samples with suspected PCR inhibition or degradation. The data was collected into a final report with discussions and conclusions to the findings for submission to the National DNA Indexing System (NDIS) Approval Board for authorization to use forensic DNA genotypes generated using MiniFilerTM in national DNA databases.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 Examining the skin and workplace microbiome following the return to the University of North Texas Health Science Center after self-isolation(2021-08) Khichi, Ophelia J.; Allen, Michael S.; Zhang, Yan; Zascavage, Roxanne R.; Rosales, ArmandoThe human skin microbiome contains trillions of microbiomes that colonize the human body. It is unknown how social distancing and an increase in handwashing due to the COVID-19 pandemic affect the bacterial communities that reside on the human hands & fomites. In this research, bacterial swabs from individual's dominant hand, forearm of their dominant hand, and a fomite from the workplace environment were taken, and the resident microflora, i.e., the skin microbiome, was investigated. The DNA from the samples were extracted and amplified by PCR. Samples were then pooled for sequencing of the V4 region of the 16S ribosomal RNA gene using Illumina's MiSeq platform and subsequently analyzed for community composition. The results were compared against each other to determine how an individual and a fomite's microbiome shifted following their return to work. Furthermore, the results were used to determine if individual's microbiome became more similar to each other as they returned to work in the same building.Item Extraction and STR Amplification of HAEIII Restriction Cut, Membrane Bound Human DNA(2005-07-01) Andrews, John S.; Joseph Warren; Arthur Eisenberg; John PlanzThe use of DNA in forensics has become widely accepted since its introduction into the field in 1985 with Restriction Fragment Length Polymorphisms (RFLP) by Alec Jeffreys (1). RFLP techniques were utilized in the forensic DNA community until the mid 1990s when less labor-intensive PCR-STR techniques became available. During the transition from RFLP technology to PCR based STR technology a method for comparing RFLP profiles to that of STR profiles was not developed. Currently there have been no published studies where STR profiles have been obtained from membrane-bound, restriction cut human DNA. The only way to compare RFLP profiles to STR profiles would be to obtain STR profiles from the bound restriction cut DNA left on the nylon membranes. Since the shift in technology from RFLP to PCR-STR most labs, including the FBI, have stopped RFLP analysis as of the year 2000 (4). Today many unsolved cases exist that utilized RFLP technology. Due to the nature of RFLP analysis many times all of the biological sample must be consumed in order to obtain an RFLP profile. When this occurs, there is no longer biological sample left for future testing. In these instances the only DNA left from the case is restriction cut and bound to nylon RFLP membranes. The only chance of determining the STR profile of the source of the biological sample found at the crime scene would be to remove the membrane bound DNA and obtain an STR profile. The experimental hypothesis of this study is that DNA can be recovered from nylon membranes and interpretable STR results can be obtained. The use of multiple STRs are highly discriminatory being able to generate rare DNA profiles possessing a discriminatory power of in many times that of the earth’s population. Due to this discrimination power, profiles are able to individualize the source of a biological sample and aid in criminal investigations. If STR profiles could be obtained from old RFLP membranes numerous cold cases could be reopened and reinvestigated. The STR profiles obtained from the RFLP membranes could be placed into the Combined DNA Indexing System (CODIS). CODIS blends forensic science and computing software into a tool for solving violent crimes. Through CODIS, STR profiles can be entered into the database and searched against possible suspects at the local, state, and national level. Obtaining STR profiles from RFLP membranes would allow for the comparison of these profiles to those found in CODIS for a possible suspect. This project will employ methods to try and obtain an STR profile from Hae III restriction cut DNA bound to Magna Graph membranes. Attempts will be made to obtain STR profiles through direct amplification off of the membrane with PowerPlex 16 and separation on the Avant 3100 equipped with GeneMapper ID. Methods will also be utilized to remove the bound DNA from the membrane prior to amplification and separation. Removal of the bound DNA from the membrane will involve physical means, as well as, the use of various extraction chemicals. If a technique is found successful at removing DNA from Magna Graph membranes, then the technique will be applied to true RFLP membranes donated by the UNTHSC.Item Internal Validation of the AmpFlSTR® Identifiler™ PCR Amplification Kit at The Forensic Testing Laboratory, Inc.(2009-08-01) Lowe, Cheryl M.; Warren, Joseph E.Prior to being implemented into forensic casework at a laboratory, a new method or product used in forensic DNA analysis must be validated internally within that laboratory. An internal validation of the AmpFlSTR® Identifiler™ PCR Amplification Kit at The Forensic Testing Laboratory was conducted, and it consisted of a sensitivity study, mixture study, non-probative case sample study, precision study, and a contamination study. The sensitivity study served to determine the target amount of input DNA to be added to each reaction, and the mixture study demonstrated how sensitive this kit is in detecting mixed source samples. The non-probative case sample study simulated various types of samples that may occur in casework. The results of each study are satisfactory for an internal validation, and the AmpFlSTR® Identifiler™ PCR Amplification Kit may now be used in forensic casework, since the kit was proven to be reliable, robust, and reproducible.