Validation of the Powerplex 16 STR System at the Harris County Medical Examiners Office




James, Donovan O.


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The 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)