The assay developed uses post-qPCR melt-curve analysis to detect the presence of double-stranded amplicon products from the targeted STRs (D5S818 and D18S51). The primary goal was to directly integrate this melt-curve assay into existing commercially available qPCR human DNA quantitation kits so as to accurately assign the sample to either a single-source geno-group or a geno-group that is typical of a mixed sample. The researchers examined two qPCR platforms, the Rotor-Gene Q and the more commonly used ABI 7500, along with several analytical approaches for the resulting melt-curve data classification: 1) use of a commercially available principal component analysis (PCA)-based melt-curve analysis software; 2) development and use of linear discriminate analysis (LDQA) code written for R statistical software; and 3) development and use of a novel support vector machine (SVM) software tool. After assessment of the tested qPCR platforms, selection of the best statistical approach, and integration of the melt-curve assay into a commercially available quantitation kit, the newly developed multiplex would need to be assessed for quantitation precision, geno-grouping concordance, and reproducibility. Overall, this work produced a qPCR-based melt-curve assay for the re-screening identification of mixtures, and the assay has been demonstrated to be viable when integrated into a commercial quantification assay. Implementation of this assay into a forensic DNA laboratory will provide analysts with more information about their evidentiary samples without the need for any additional steps in the workflow; however, there are several considerations that must be addressed prior to crime lab implementation. These considerations are discussed in this report. 13 tables, 6 figures, and 18 references
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