Toward Analysis at the Point of Need: A Digital Microfluidic Approach to Processing Multi-Source Sexual Assault Samples

Abstract

Forensic case samples collected in sexual assaults typically contain DNA from multiple sources, which complicates short-tandem repeat (STR) profiling. These samples are typically sent to a laboratory to separate the DNA from sperm and non-sperm sources prior to analysis. Here, the automation and miniaturization of these steps using digital microfluidics (DMF) is reported, which may eventually enable processing sexual assault samples outside of the laboratory, at the point of need. When applied to vaginal swab samples collected up to 12 h post-coitus (PC), the new method identifies single-source (male) STR profiles. When applied to samples collected 24-72 h PC, the method identifies mixed STR profiles, suggesting room for improvement and/or potential for data deconvolution. In sum, an automated, miniaturized sample pre-processing method for separating the DNA contained in sexual assault samples is demonstrated. This type of automated processing using DMF, especially when combined with Rapid DNA Analysis, has the potential to be used for processing of sexual assault samples in hospitals, police offices, and other locations outside of the laboratory.

Keywords: differential digestion; digital microfluidics; rapid DNA analysis.

Figure 1

Differential digestion on DMF. a) Schematic (partly created with BioRender.com) showing the DMF‐assisted differential digestion protocol (DMF‐DD). First (in steps that are implemented off‐chip), non‐sperm cells are selectively lysed using non‐sperm lysis buffer (that does not contain DTT), and then the sperm cells are pelleted by centrifugation. Second (in steps that are implemented on‐chip), the resuspended sperm pellet is loaded into the DMF chip and processed. Third, the male fraction of DNA is purified, amplified and then characterized by STR analysis automatically using ANDE^TM 6C Rapid DNA System using the ANDE I‐Chip. b) Images (1) to (6) are frames from Video S1 (Supporting Information) demonstrating a portion of the steps that are automated by DMF (with dyes included for visualization). The following steps are performed automatically by activating electrodes in succession: 1) move sample (pink) from reservoir, 2) merge DNase (green) with the sample, 3) mix by moving in circles continuously for 15 min at 37 °C, 4) add EDTA (red) and mix for 10 min at 65 °C, 5) add DTT and proteinase K (blue) and mix for 15 min at 56 °C, and 6) collect DNA for analysis.

Figure 2

Representative electrophoretic analysis of sample 1, a vaginal swab collected 1 h post‐coitus, generated using standard laboratory analysis techniques. a) Partial electropherogram of fluorescence intensity (red trace) as a function of fragment length (bp) of the non‐sperm fraction. Labels above the plot (green filled boxes) indicate which markers the detected peaks correspond to, including the sex‐typing marker Amelogenin (“Am”) and STR loci D5S818 and FGA. Labels below the plot (red outlined boxes) indicate the number of repeats for STR loci or X/Y designation for sex typing, as determined using GeneMapper^TM ID‐X genotyping software (Applied Biosystems), as well as the peak height in rfu. b) Partial electropherogram of sperm fraction processed by manual DD. c) Partial electropherogram of sperm fraction processed using DMF‐DD.

Figure 3

Representative electrophoretic analysis of sample 7, a vaginal swab collected 12 h post‐coitus, by standard laboratory analysis techniques and Rapid DNA analysis. a) Partial electropherogram of fluorescence intensity (red trace) as a function of fragment length (bp) of the DMF‐DD sperm fraction, generated using standard laboratory analysis techniques. Labels above the plot (green filled boxes) indicate which markers the detected peaks correspond to, including the sex‐typing marker Amelogenin (“Am”), and the STR loci D5S818 and FGA. Labels below the plot (red outlined boxes) indicate either the number of repeats for STR loci or X/Y designation for sex typing as determined using GeneMapper^TM ID‐X genotyping software (Applied Biosystems), as well as the peak height. b) Partial electropherograms of fluorescence intensity (blue, yellow, and purple traces) as a function of fragment length of the DMF‐DD sperm fraction generated using the ANDE^TM 6C system. Labels above the three plots (gray filled boxes) indicate the markers the detected peaks correspond to, and labels below the plot (gray filled boxes) indicate either the number of repeats for STR loci or X/Y designation for sex typing. Blue, yellow, and purple boxes (and arrows) indicate loci in a) that correspond to loci in b): Am, D5S818, and FGA, respectively.

Figure 4

STR Quality Analysis. PCR product characterization for samples 4 to 7 (collected 1, 3, 6, and 12 h post‐coitus), using the electropherograms generated by conventional laboratory analysis. Heterozygous peak height balance (black diamonds) was calculated by dividing the smaller peak height of the two alleles by the larger one. Stutter peak (red diamonds) was calculated by dividing the stutter height by the larger of the peak heights for the corresponding allele at the same locus.