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Current extraction methods employed by the Columbus Police Forensic Services Center use incubation times that can exceed two hours and require subsequent purification on robotic instrumentation. Promega’s Casework Direct kit allows for the rapid processing of casework samples with no subsequent purification of the lysate required prior to STR amplification. Typically, the processing of sexual assault cases involves numerous differential extractions that are time-consuming and require hands-on sample manipulation and tube transfers. The Casework Direct extraction includes a thirty-minute incubation with no tube transfers. Samples are extracted in 300ul of Casework Direct Reagent, with the addition of 1.5ul of 1-Thioglycerol as a reducing agent. The lysate can be further quantified and amplified using existing instrumentation and protocols. Samples needing differential extractions based on the quantification data can be identified and extracted accordingly. The Casework Direct kit was originally evaluated strictly as a Y-screening tool. However, it was observed during the first few data sets that it consistently yielded higher DNA quantification values much faster and easier compared to the current extraction method. Further evaluation of high and low level DNA samples was initiated to consider using Casework Direct as the main extraction method for all samples (excluding hairs). Casework data has shown this method to yield better amplification of minor alleles at higher [Auto]/[Y] ratios. This has reduced the number of sexual assault samples requiring a differential re-extraction. Current validation work is being performed on possible clean-up methods to help remove impurities that may affect downstream processing for certain samples. These clean-up methods include the Maxwell®, AutoMate ExpressTM, and QIAGEN EZ1 extraction robots, and the QIAquick PCR purification kit. This may help reduce the number of samples that would require re-extraction and allow the use of Casework Direct on more low-level touch DNA samples.
Current extraction methods employed by the Columbus Police Forensic Services Center use incubation times that can exceed two hours and require subsequent purification on robotic instrumentation. Promega’s Casework Direct kit allows for the rapid processing of casework samples with no subsequent purification of the lysate required prior to STR amplification. Typically, the processing of sexual assault cases involves numerous differential extractions that are time-consuming and require hands-on sample manipulation and tube transfers. The Casework Direct extraction includes a thirty-minute incubation with no tube transfers. Samples are extracted in 300ul of Casework Direct Reagent, with the addition of 1.5ul of 1-Thioglycerol as a reducing agent. The lysate can be further quantified and amplified using existing instrumentation and protocols. Samples needing differential extractions based on the quantification data can be identified and extracted accordingly. The Casework Direct kit was originally evaluated strictly as a Y-screening tool. However, it was observed during the first few data sets that it consistently yielded higher DNA quantification values much faster and easier compared to the current extraction method. Further evaluation of high and low level DNA samples was initiated to consider using Casework Direct as the main extraction method for all samples (excluding hairs). Casework data has shown this method to yield better amplification of minor alleles at higher [Auto]/[Y] ratios. This has reduced the number of sexual assault samples requiring a differential re-extraction. Current validation work is being performed on possible clean-up methods to help remove impurities that may affect downstream processing for certain samples. These clean-up methods include the Maxwell®, AutoMate ExpressTM, and QIAGEN EZ1 extraction robots, and the QIAquick PCR purification kit. This may help reduce the number of samples that would require re-extraction and allow the use of Casework Direct on more low-level touch DNA samples.
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