Browsing by Subject "PCR"
Now showing 1 - 13 of 13
- Results Per Page
- Sort Options
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 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 Molecular Techniques Using a Synthetic Mitochondrial Genome(2014-05-01) Koenig, Jessica L.; Rhonda RobyThe mitochondrion is responsible for the bulk of cellular energy production through the process of oxidative phosphorylation. The mitochondrial genome (mtGenome) is subject to a high mutation rate due to its proximity to reactive oxygen species produced in energy production. Over 250 pathogenic mutations have been characterized, and studies have demonstrated mtDNA variations at the cellular, tissue, and individual level. Some of the characterization techniques include long range PCR and sequencing. Sanger sequencing has been the gold standard, but next-generation sequencing technologies are now available. These methods may be evaluated using synthetic DNA of known base composition. This project utilizes the first synthetic mtGenome to optimize a LR PCR protocol and evaluate sequence quality using Sanger, MiSeq System, and Ion Personal Genome Machine System sequencing platforms.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 Human Hair Shaft Volume and Mitochondrial DNA Recovery(2007-08-01) Kreikemeier, Melissa A.; Arthur Eisenberg; Joseph Warren; John PlanzKreikemeier, Melissa A., Human Hair Shaft Volume and Mitochondrial DNA Recovery. Master’s of Science (Biomedical Science, Emphasis in Forensic Genetics) August, 2007, 54 pp., 6 tables, 26 figures, References, 45 titles. The goal of this study was to determine if there was a relationship between hair volume and amount of amplified mtDNA for all head hair samples, as well as among the three major ethnic groups, different hair colors, and different donors. This relationship could be used to determine how much of a hair sample is needed for extraction so that enough mtDNA PCR product is obtained, while preserving forensic hair samples and avoiding unnecessary consumption of evidence. Amplification success rates were also calculated for each of these categories to determine if the findings coincided with previously published literature.Item Molecular Cloning and Regulation of Expression of an NK Cell Receptor(2001-07-01) Medina, Miguel Angel; Porunelloor Mathew; Rafael Alvarez-Gonzales; Neeraj AgarwalNatural killer (NK) cells are large granular lymphocytes derived from bone marrow. They form the first line of defense against virally infected and tumor cells. Unlike B and T cells, they are not MHC restricted therefore do not require prior antigen stimulation (1-4). NK cell functions include producing various cytokines such as interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and granular-macrophage colony stimulating factor (GM-CSF) and cytotoxicity (5,6). A number of cell surface molecules have been identified, cloned and characterized that modulate NK cell recognition and activation by target cells (1). Most of these molecules are also expressed on other leukocytes. NK cell function is regulated by the balance of the positive and negative signaling through these receptors (3, 7-10). In the past attention has primarily focused on major histocompatibility complex (MHC) recognizing receptors that are mostly inhibitory (11). It is through these inhibitory receptors that levels of MHC molecules and associated peptides are monitored. Cells that have lost the expression of MHC class I molecules or have altered peptides-class I complexes are not able to transmit an inhibitory signal to NK cells and are consequently killed. Members of the CD2 subset of receptors play a major role in lymphocyte functions and do not recognize MHC molecules. The signaling lymphocyte activation molecule, SLAM (CD150), a member of the CD2 subset, is expressed on T cells and B cells. SLAM regulates T cell activation and production of immunoglobulins by B cells (12,13). 2B4 is a member of the CD2 subset and is expressed on NK cells as well as other leukocytes (14, 15). 2 B4 is a surface molecule implicated in the activation of NK cell-medicated cytotoxicity (15-17). Human 2B4 is a 60-70 kDA glycoprotein surface molecule found on all NK cells and a small subset of T cells that exhibit NK-like activity. CD48 has been identified as the high affinity ligand for 2B4 and implicates a broader role for 2B4 in immune regulation (18, 19). Recent reports have demonstrated the importance 2B4 and the functional role 2B4 plays in immune regulation. In X-linked lymporoliferative (XLP) disease NK cells can not be activated through surface 2B4 (20-23). The molecular adaptor protein, SLAM-associated protein or SH2 domain containing adaptor molecule (SAP/SH2D1A) is associates with cytoplasmic tail of 2B4 or SLAM (24, 25). Defective signaling via 2B4 and SLAM may contribute to the pathogenesis of X-linked lymphoproliferative disease due to mutations in SAP. The cytoplasmic domain of 2B4 contains four novel tyrosine motifs (TxYxxV/I) (14, 15). SLAM, a close relative of 2B4, also contains these novel tyrosine motifs. The signaling mechanism for 2B4 remains unclear. Along with other members of the CD2 subset 2B4 also localizes to chromosome 1. The genes that encode the CD2 family of receptors are locatedon human chromosome at 1q21-24 (24, 26-30). The murine genes for 2B4, CD48, Ly49, Ly108, and CD84 are located on the syntenic region of the long arm of the chromosome 1 (30-33). The exon arrangement for 2B4 is consistent with other CD2 subset members and consists of an exon per domain for the leader sequence, V-like domain, C2-like domain, and the transmembrane domains (27, 34-37). Differential exon usage leads to splice variants of the receptors, which complicates understanding the functional relevance between the cytoplasmic domains between receptors. Both murine 2B4 and SLAM demonstrates splice variants that alter the number of novel tyrosine motifs within the cytoplasmic domains (14, 34, 38). The murine 2B4 gene consists of 9 exons with one exon dedicated to each leader sequence, V-like, C2-like, and transmembrane domains. The total gene size is approximately 27 kilobases with the first intron consisting of 16 kilobases. Variable exon usage gives rise to two isoforms of 2B4, 2B4-L and 2B4-S, in the mouse (38). Four exons encode the 2B4-L cytoplasmic domain, giving rise to four tyrosine motifs. 2B4-S is identical to the 5’end of 2B4-L, differing only at the 3’ end in a portion of the cytoplasmic domain and the 3’untranslated sequence. 2B4-S is the product of the same first five exons in 2B4-L with the usage of a novel exon at the C-terminal. Although splice variants exists there Is no direct biochemical evidence to support their expression. In vitro analysis of the m2B4 variants suggest potential signaling differences. Murine 2B4 variants and mutants were transfected into a rat NK cell line, RNK-16. Interestingly, the two forms of 2B4 had opposing functions (39). Murine 2B4 is expressed on all NK cells, a subset of T cells, dendritic epidermal T cells, and monocytes (40). Expression levels of 2B4 can be elevated by incubation with interleukin-2 (IL-2). Engagement of 2B4 can be elevated by incubation with interleukin-2 (IL-2). Engagement of 2B4 with anti-2B4 monoclonal antibody (mAb) causes secretion of interferon-γ, increased 2B4 expression, and elevated cytotoxicity (41). Characterization of how 2B4 and its related receptors are expressed is critical to the understanding not only the receptors’ biology but also NK cell biology. My first project will focus on mastering the techniques involved in the isolation and characterization of genes. Previously two genomic clones were isolated from 129 Sv/J mouse liver, 531 and 532. The first clone, 531, has been fully characterized and revealed to be 2B4. 532 has been partially characterized and revealed to the related form of mouse 2B4. In order to determine the function of 532 on mouse NK cells, 532 cDNA has to be isolated. I attempted to isolate 532 cDNA through PCR using previously isolated clones from the BALB/c cDNA library. My next aim was to isolate 532 genomic DNA for automated sequencing. I used this data to design primers specific for 532 and isolate the 532 cDNA through RT-PCR. 532 will be the topic discussed in chapter 2 and chapter 3 will discuss the 2B4 activated sequencing. I used this data to design primers specific for 532 and isolate the 532 cDNA through RT-PCR. 532 will be the topic discussed in chapter 2 and chapter 3 will discuss the 2B4 activated response molecule. The final portion of my thesis will focus on the isolation of the 2B4 activated response molecule (2ARM). Human peripheral blood NK cells were isolated incubated with interleukin-2 or C1.7. C1.7 is a monoclonal antibody that specifically recognizes human 2B4. RNA was extracted from these NK cells at various time points and used for RT-PCR to monitor the expression levels of 2B4. Aside from the expression of human 2B4, the expression of a 160 base pair transcript was also detected. Sequencing analysis revealed this transcript to be novel. I screened a human NK cDNA library constructed by Dr. J. Houchins (R & D System, Minneapolis, MN) using this 160 base pair transcript as a probe. Upon isolation of 2ARM cDNA, functional analysis can be performed to determine its role on human NK cells.Item Obesity Genetics: The Prevalence of DRD2, DAT1 and DBH Genes in the Obese Individual(1998-08-01) Davis, Karla R.; Eisenberg, Arthur; Agarwal, Neeraj; Sherman, MarkDavis, Karla R., Obesity Genetics: The prevalence of DRD2, DAT1 and DBH Genes in the obese individual. Master of Science (Biomedical Sciences), August, 1998, 106 pp., 3 tables, 14 illustrations, reference, 44 titles. Obesity has been presented in research literature as a polygenic or multiple gene disorder. Currently, 3 genes have been associated with obesity, dopamine receptor D2 (DRD2), dopamine transporter (DAT1), and dopamine beta hydroxylase (DBH). The primary objective of this study is to analyze the DRD2, DAT1 and DBH genes to determine if a correlation exists between certain allelic variations of these 3 genes and the body mass index of obese individuals. We have developed an assay for the DRD2, DAT1 and DBH genes, utilizing polymerase chain reaction (PCR) technology. Within the DRD2 gene, 2 allelic variants have been identified, the A1 and A2 alleles. The A1 allele consists of a 310 bp fragment in which the Taq 1 restriction site has been deleted. The A2 allele consists of 180 bp fragment and a 130 bp fragment. The presence of the A1 allele after enzyme digestion has shown a strong correlation to obesity in prior studies. With respect to the DAT1 gene, a VNTR of 40 bp’s has been correlated to other disorders within the ‘reward deficiency syndrome’. The fragment length identified most often is 440 or 480 bp, with 480 as the primary fragment in obesity. The DBH gene is similar to the DRD2 in that it also contains a Taq I restriction. Two allelic variants are also identified, B1 and B2. The B1 allele contains no Taq I site and produces a 316 bp fragment while the B2 does cleave, exhibiting an 86 bp and a 230 bp fragment after enzyme digestion. The presence of one or more of the aberrant alleles could be associated with and a predisposing factor to obesity.Item Testing Minimum Ultraviolet Light Exposures to Effectively Remove Contaminating DNA for Use in Forensics(2005-08-01) Kanaly, Angela Catherine; John Planz; Joseph Warren; Arthur EisenbergThis study introduces a solar lamp UV light source, for the purpose of removing contaminating DNA in direct relation to forensic testing. The study attempts to demonstrate what level of decontamination occurs from sun lamp exposure at given time intervals of exposure, set at distances from the lamp, and for different types of biological samples. A FS-40 solar lamp was used to irradiate samples of amplified DNA and cellular samples at distances of 5 cm, 10 cm, and 60 cm from the source, with varied exposure times of 15 min, 30 min, 3 hrs, 6 hrs, 12 hrs and 24 hrs. Common forensic DNA typing concerns include contamination by previously amplified DNA products or from transfer of cellular material onto testing materials. Samples exposed included dried PCR products amplified by AmpFlSTR COfiler kit, dried whole blood, and dried saliva. An organic extraction of the blood and saliva samples isolated any remaining genomic DNA. Control blood and saliva samples were quantitated for accurate DNA concentration. All samples were then amplified by AmpFlSTR COfiler kit and analyzed on an ABI 310 Genetic Analyzer, along with an unexposed control PCR product, blood, and saliva samples, reagent blank run alongside each PCR product, blood, and saliva series, and positive and negative PCR controls. Fragment analysis data was analyzed by GeneScan and Genotyper software to obtain any detectable genetic profile from the samples. This experimental design mimics a true forensic casework scenario by following a routine chain of procedures used widely throughout the field. The current standard in forensic DNA testing measures short tandem repeats (STRs), which vary significantly in length between individuals. There are thirteen loci used by the Combined DNA Index System (CODIS), the national DNA index managed by the FBI Laboratory. All thirteen loci are typed in a typical DNA test, with the AmpFlSTR COfiler kit amplifying seven of these loci. For the purposes of this study, successfully decontaminated PCR products, blood, and saliva samples would show no detectable genotype at any of the seven loci. Other DNA testing, such as mitochondrial DNA analysis from hair, bone or teeth, or very low copy number DNA from a small number of cells, require extreme caution to avoid contamination, as these tests have increased sensitivity over standard STR testing. The level of decontamination detected through UV exposure in this study would not provide sufficient information for application to the more sensitive techniques.Item Validation of Four Multiplex SNP Panels for Forensic DNA Testing: An Assessment of the Sensitivity and Reproducibility of the SNaPshot Primer Extension Assay(2003-05-01) Dutton, Kristi R.; John Planz; Arthur Eisenberg; Joseph WarrenOrchid Cellmark provided four proprietary multiplex SNP primer panels, each of which has been developed to identify 10-12 SNPs using a modified version of the SNaPshot Mutliplex protocol. The commercially available STR typing kits routinely used in forensic testing require an input of between 0.5 to 2ng of DNA. Orchid Cellmark has suggested using 2ng of DNA with each of their multiplex SNP primer panels. However, preliminary data has indicated that as little as 100pg of DNA can yield results with many of the SNP markers. Several methods of SNP detection exist. This project relied on the use of multiplex SNP extension primers in conjunction with the ABI SNaPshot Multiplex Kit (Applied Biosystems, Foster City, CA) to identify SNPs in the nuclear genome. Analysis of 50 or more SNPs would be very laborious if single-tube polymerase chain reaction (PCR) was used for sample testing. Multiplexing SNP primers to include 10-12 per reaction tube will increase the throughput of SNP analysis. The cost of analysis can also be reduced using multiplexes since the amount of reagents per SNP is decreased. This project determined if the SNaPshot extension assay used in conjunction with the Orchid Cellmark multiplex panels could accurately detect SNPs at quantities less than 2ng DNA on a capillary electrophoresis (CE) platform.Item Validation of the Powerplex 16 STR System at the Harris County Medical Examiners Office(2006-07-01) James, Donovan O.; Arthur Eisenberg; Joseph Warren; John PlanzThe amplification and detection of extracted DNA are essential steps in the processing of forensic case work for DNA typing. Over the years the forensic community has strived to improve the techniques used for DNA typing. The methods used for DNA typing have advanced significantly due to the use of Polymerase Chain Reaction (PCR) technique developed in 1985 by Kary Mullis [1]. This technique provides the ability to amplify minute amounts of DNA at specific regions of interest called short tandem repeats (STR’s). The PCR reaction is well adapted for DNA amplification because it is sensitive, rapid, and has the potential to analyze degraded samples. The evolution of DNA typing methods has progressed from our ability to analyze one DNA region of interest at a time with the restriction fragment length polymorphism (RFLP) procedure to typing several STR markers using multiplexing PCR methods. A total of 13 specific STR markers were selected by the FBI in order to standardize analysis of DNA for use in a nation wide database. The Combined DNA Index System (CODIS) is the database developed to provide the comparison between crime scene evidentiary samples and known samples from previously convicted criminals. Commercial kits are available which provide forensic laboratories the ability to amplify the 13 core STR loci required for the CODIS database. One such amplification system is the AmpFlSTR Profiler Plus PCR amplification kit (Applied Biosystems, Foster City, CA). The AmpFlSTR Profiler Plus kit amplifies 9 of the 13 CODIS loci plus the Amelogenin sex typing marker. This kit is used in conjunction with a sister kit, the AmpFlSTR COfiler PCR amplification kit which amplifies the additional 4 core loci along with 2 overlapping loci (D3S1358 and D7S820) and Amelogenin. Promega Corporation (Madison, WI) has developed the PowerPlex 16 PCR multiplex system which amplifies the 13 core STR loci plus two additional STR systems and Amelogenin in a single PCR reaction. The Harris County Medical Examiners (HCME) Office located in Houston, Texas has validated the AmpFlSTR Profiler Plus and COfiler PCR Amplification system for use in their DNA laboratory. The HCME DNA laboratory was interested in incorporating the newly improved PowerPlex 16 PCR system (Promega Corp., Madison WI) for DNA amplification and detection purposes. The use of the PowerPlex 16 system would provide a single amplification system while maintaining the sensitivity and robustness required for forensic DNA testing. Prior to implementation in casework, the HCME laboratory was required to perform internal validation experiments to assess the performance and limitations of the system in the laboratory. The study undertaken at HCME was designed to fulfill the requirements mandates by national standards issued by the Director of the FBI and guidelines issued by the Technical Working Group on DNA Analysis Methods (TWGDAM)