Browsing by Subject "Applied Biosystems"
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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 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 Internal Validation of Applied Biosystems Quantifiler Duo DNA Quantitation Kit For Use in Fornesic Casework(2008-08-01) Klein, Cindi Leann; Arthur Eisenberg; Joseph Warren; John PlanzKlein, Cindi Leann. Internal Validation of Applied Biosystems Quantifiler Duo DNA Quantification Kit for Use in Forensic Casework. Master of Science (Forensic Genetics), August 2008, 48 pages, 19 figures, 4 tables, 10 references. Applied Biosystems (Foster City, CA) has recently developed a new DNA quantification kit, Quantifiler Duo, which simultaneously quantitates the total amount of amplifiable human and male DNA present in a sample. The kit is a multiplex real-time PCR system based on the 5’ nuclease assay and Taqman probe-based technology. An internal validation was performed at the Harris County Medical Examiner’s Office using Quantifiler Duo and was comprised of six studies: sensitivity, reproducibility, cross-contamination, precision, mixture, and stability. The preliminary results show an increased sensitivity from the currently used Quantifiler Human and Quantifiler Y Human Male DNA Quantification kits. The accuracy and precision of the Quantifiler Duo kit was also comparable or superior to that of the current kits.