A modified direct PCR amplification method using the GlobalFiler™ PCR Amplification Kit on bloodstains collected using microFLOQ™ direct swabs

Abstract

The standard forensic DNA analysis workflow typically encompasses DNA extraction, quantification, STR-PCR amplification, and CE detection. A direct PCR amplification method eliminates the extraction and quantification steps, shortening the turnaround time for DNA profiling. However, a limitation to the direct PCR amplification method lies in its inability to allow for additional PCR amplification on the same sample. We found that replicate PCR amplifications can be afforded with the following modification to the direct PCR amplification method: after sample collection, the microFLOQ™ Direct swabs were incubated in low TE buffer prior to PCR amplification of the lysate. With replicate amplifications, the impact of stochastic effects during STR-PCR amplification on DNA profile interpretation would be reduced. Additionally, adjustments can be made to template volumes in subsequent amplifications, preventing oversaturated PCR reactions. Our results showed that this modified direct amplification method gave comparable median peak heights, allele recovery and intra-locus peak-height-ratio to those of the standard workflow, while maintaining the advantage of minimal evidence consumption.