Evaluating DNA Sample Degradation With a Quantitative Gender Typing End-Point PCR Multiplex

Abstract

DNA sample degradation is a common problem encountered in forensic DNA casework. In some instances the severity of sample degradation prevents the inclusion of an evidentiary profile into the FBI CODIS database. In this study an end-point PCR quantitative assay is assessed to determine if human DNA targets of differing size can be used to predict DNA sample degradation in STR profiling. Primer sequences from a small male-specific target (SRY) and a large human target (Amelogenin) are multiplexed. These targets estimate the respective total-human and male-only quantities of sample DNA based on comparison to a standard curve prepared from known quantities of human DNA. The quantitative estimate of the small target versus the quantitative estimate of the large target can be compared in male samples to form a ratio. This ratio may indicate the probable amplification performance of large-target STR loci multiplexed in HID typing kits. A method was developed here to degrade DNA in a controlled manner for analysis with this quantitative assay. The resulting quantitative ratios were compared to HID ratios developed from the amplification performance of small and large CODIS core loci. In addition, a selection of male reference samples was also examined to evaluate the correlations of quantitative ratios and HID ratios obtained in actual casework. Regression analysis of the performance of the small and large targets amplified in the quantitative assay demonstrated a significant difference (p < 0.001) between both targets through the course of the in vitro degradation experiment. These results indicate that in degraded samples the small target will outperform the large target in the quantitative assay. Assessment of the quantitative ratios demonstrated significant degradation (p < 0.001) at and above the t=60 minute timepoint in the successive degradation of samples. This experimental model was applied to the quantitative and HID typing results obtained from male reference samples in actual casework. Statistical assessment of these samples indicates a strong correlation (r = 0.7478, p < 0.001) between the quantitative ratios and the HID ratios. This confirms the ability of the quantitative assay to assess large-target amplification performance via a ratio, which predicts the trend of sample degradation observed in HID profiling.