Inhibition of DNA amplification caused by metal in extracted bloodstains and in direct amplification

Abstract

Copper is a metal that is commonly found in many evidence types, such as weapons, and household items, that are often retrieved from crime scenes. We used copper and brass sheets, cartridge cases, and metal décor items as substrates for deposition of blood to study inhibition induced by metals. Another objective was to compare the efficiency of STR profiling following extraction, quantification, amplification, and capillary electrophoresis with a direct amplification method which only requires amplification and capillary electrophoresis. Samples that were designated for extraction, were collected, extracted, and purified using the Qiagen EZ1 DNA Investigator® Kit. Direct amplification was performed using Copan microFLOQ® Direct Swabs to collect minute amount of blood. Complete STR profiles were obtained from 54% of the extracted samples and from 88% of directly amplified samples. Substrate type and amplification method (two-way ANOVA, F(5, 131) = 3.59, p = 4.50 × 10−3) impacted profile completeness but collection time did not (one-way ANOVA, F(1, 131) = 0.017, p = 0.90). Composition of the substrates was determined with scanning electron microscopy (SEM), and inhibition caused by copper was evaluated for each substrate. When substrate composition was analyzed in combination with amplification method, a substrate’s copper percent was not associated with increased allelic dropout (one-way ANOVA, F(1, 176) = 0.70, p = 0.40) but amplification alone was statistically significant (one-way ANOVA, F(1, 176) = 40.64, p = 1.56 × 10−9). Using the Copan microFLOQ® Direct Swabs, inhibition induced by metal observed was not as pronounced. In addition, less variability in profile completeness was detected, profiles were generated within a very short period, and only minute amount of bloodstain was needed, allowing for additional analysis of the same samples. Our results directly quantify the effect of copper composition on DNA profiling and suggest that while copper percentage is an observational contributor to allele dropout it is not wholly responsible for these events.