Browsing by Subject "STR analysis"
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Item Authentication of Human Cell Lines and Detection of Human Cell Line Contamination by Cost-Effective Direct Amplification of DNA(2014-12-01) Vemireddy, Vikram Reddy; Patricia A. GwirtzHuman cell lines are extensively used in scientific research. Cell line contamination can occur anytime during a research project due to exposure to another human cell line. Thus, authentication of human cell lines is recommended multiple times during research. The Short Tandem Repeat (STR) analysis method has been used to authenticate human cell lines. STR is a complex process involving several steps, such as DNA extraction and purification. To make this identification method simple and quick, manufacturers have been manufacturing kits that can amplify the DNA directly from cells spotted on a storage medium without DNA extraction and purification. Many laboratories offer the direct cell line authentication service at a high cost due to these laboratories using expensive kits and storage media. In this practicum study, the Human ID Bloodstain Cards (GE Healthcare Life Sciences, Piscataway, NJ) and the GenePrint® 10 System Kit (Promega Corp., Madison, WI) were used to establish a cost-effective direct STR amplification method. Three concentrations of DU-145 cell suspension solutions (5000 cells/μL, 12,500 cells/μL, and 25,000 cells/μL) were spotted onto a Human ID Bloodstain Card, and two card punches of 1.2 mm diameter were taken from each concentration for analysis. The American Tissue Culture Collection (ATCC, Manassas, VA) established the ASN-0002 standard for cell line authentication, which recommends amplification of a minimum of eight autosomal loci and sex determining loci (Ameleogenin). The results showed that the STR profiles obtained from the Human ID Bloodstain Card samples using the GenePrint® 10 System kit met the ASN- 0002 standards.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 Validation of Bodeplex 3 for Typing Small Amplicons and Low Copy Number DNA(2003-07-01) Chahrour, Maria H.; Arthur Eisenberg; John Planz; Joseph WarrenChahrour, Maria H., Validation of BodePlex3 for typing small amplicons and low copy number DNA. Master of Science (Forensice Genetics), July, 2003. 76 pp., 14 tables, 6 figures, references, 25 titles. The BodePlex 3 multiplex is a sensitive mini-STR typing system for analyzing highly degraded and low copy number (LCN) DNA. It allows for the typing of smaller amplicons and decreased quantities of DNA template, thus enhancing the sensitivity of STR analysis of compromised samples. The present study validated BodePlex 3 multiplex system on the ABI PRISM 3100 Genetic Analyzer to be used at The Bode Technology Group for forensic DNA analysis of small amplicons and LCN DNA. Validation experiments were performed according to the DNA Advisory Board (DAB) guidelines. Performance of the multiplex was accurate, reliable and reproducible. The results indicated that the typing system is highly specific for human DNA, sensitive for detecting profiles of LCN DNA, and is capable of resolving mixtures to a certain extent. In addition, this project outlined possible limitations that must be considered for successful use and interpretation of BodePlex 3 DNA profiling results.