Browsing by Subject "Promega"
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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 Internal Validation Study of Promega's Powerplex Y System(2004-08-01) Donovan, Erin W.; Joseph Warren; John Planz; Arthur EisenbergThe expectations of this validation study and results obtained are similar. For the sensitivity study, Promega documents the optimal amount of DNA to be added to the PCR reaction 500pg to lng, (11) which is consistent with the results of this validation study. For the mixture study of male and female DNA Promega claims up to a concentration [greater than] 100X female DNA compared to male DNA is acceptable with no effects. This was the case in the validation study, which had a proportion of 1:50 with no effect. The substrate study showed leather to inhibit the PowerPlex Y System which has been see to happen in other DNA testing (11). The environmental study test showed humidity effects the results of the PowerPlex Y system. Also this frequently has been in other DNA testing due to the humidity causing DNA degradation, which results in a partial profile or no results for that sample. The results obtained from this internal validation study can be compared to other validation studies for Y chromosome specific STR multiplex systems. For example the paper “Validation and casework application a Y Chromosome specific STR multiple” written by Mechthild Prinz is comparable to that of this validation study performed (3). The Y-STR multiplex tested only four loci versus the PowerPlex Y System which tests twelve. The studies addressed in each of the validation studies consist of mixture, sensitivity, environmental, and substrate. For the male-female mixture study, the female DNA had no effect on the results up to 1:4000 ratios. These results are similar to the Powerplex Y System because no effect on the ability to obtain a full profile with a mixed sample of female and male DNA was observed. The sensitivity for the multiplex system showed allelic drop out at 125ph of DNA and optimal amount of DNA at 1-2ng. The PowerPlex Y System showed allelic drop out at 250ph and optimal amount of DNA to be 500pg-1ng. The environmental studies were similar because both showed allelic drop out for the humidity conditions. The multiplex with four loci did not use leather as a substrate, the other substrates were similar in both validation studies and reported similar results. Validation studies are frequently needed in order to validate a technique to be used in a forensic laboratory. Forensic cases are frequently under scrutiny by the judicial system. The questions due not lie in the science behind DNA testing but rather the process by which a laboratory performs the DNA test. In order to reduce the amount of questions validation studies are performed and interpretation guidelines are developed from those validation studies. Interpretation guidelines are written to direct and assist an analyst in making a final interpretation of each individual sample. The guidelines consist of the control, which must be run alongside each sample with the excepted results of each of the controls. The guidelines include types of identification for a sample such as no result, inconclusive, exclusive, and not excluded. Also DNA quantification information is included along with internal lane standard and allelic ladder guidelines. Interpretations of mixtures are included, which explains peak height ratios and how to determine a mixed sample. A calculation section is included to address the appropriate calculations to be used with the system to analyze the results. Interpretation guidelines will be pronounced based on this validation study for the University of North Texas Health Science DNA Identity laboratory.Item The Evaluation of the Maxwell 16 and the DNA IQ Casework Sample Kit for the Extraction of DNA from Forensic Samples(2007-08-01) Alkhazin, Tarig; Arthur Eisenberg; Joseph Warren; John PlanzThe Maxwell 16 (Promega Corporation, Madison, WI) is a small, self-contained instrument utilizing Promega’s DNA IQ chemistry for the automated extraction of DNA from 16 biological samples simultaneously. Currently, the Maxwell 16 is used in conjunction with the DNA IQ Reference Sample kit for the automated extraction of DNA from forensic and paternity reference samples. Promega Corporation is currently in the development of the DNA IQ Casework Sample kit with the intent of using the Maxwell 16 instrument in the extraction of DNA from forensic evidentiary samples. Modifications have been made to Maxwell 16 to allow the elution of DNA in a smaller volume (Low-Elution Volume (LEV) configuration) that when used in conjunction with the DNA IQ Casework Sample kit would optimize DNA yield from forensic casework samples. However, the limited quantity and the low quality of forensic casework samples are significant challenges facing most automated DNA extraction systems. An evaluation study was conducted to test the performance of the Maxwell 16 instrument along with the DNA IQ Casework Sample kit for processing forensic evidentiary samples. Mock evidentiary items used for the evaluation consisted of blood, semen, tissue, and touch samples that are routinely encountered in forensic casework. Prior to loading the Maxwell 16 instrument samples were first digested using the Tissue and Hair Extraction kit (Promega Corporation) designed for optimum DNA recovery. The extraction of DNA from sexual assault samples typically requires the separation of the sperm DNA deposited by the assailant from the vaginal epithelial cell DNA from the victim. Promega Corporation has developed the Differex System which utilizes a manual phase separation technique to obtain both a sperm fraction and an epithelial cell fraction. After separation and digestion, samples were then added to the DNA IQ Casework Sample kit cartridges for automated DNA purification using the Maxwell 16. For comparison, DNA was also obtained from replicate samples processed with the Differex System following the standard organic extraction method. The evaluation of the Differex system with mock sexual assault samples was conducted in order to determine if this differential extraction process can be used in conjunction with Maxwell 16 to improve case processing efficiency. To evaluate the performance of the Maxwell 16 and the DNA IQ Casework Sample kit, replicate samples were prepared and processed using UNTHSC’s standard organic extraction methodology. The DNA obtained by both methodologies was quantified using the Applied Biosystems (AB) Quantifiler Human DNA Quantification kit (Foster City, CA) and the AB 7500 Real-Time PCR system and then amplified using the PowerPlex 16 kit (Promega Corporation). The amplified DNA was analyzed using the AB 3130xl Genetic Analyzer and the resulting STR electropherograms were analyzed to obtain profiles using GeneMapper ID v3.2 (Applied Biosystems). In addition to assessing the quantity and the quality of the DNA obtained, blank cartridges were simultaneous processed with the mock forensic samples to demonstrate whether the Maxwell 16 could introduce cross-contamination between samples. The overall performance and the cost-effectiveness are the ultimate criteria to help determine the utility of the Maxwell 16 in the processing of forensic evidentiary casework samples.