Date Published: February 7, 2019
Publisher: Public Library of Science
Author(s): Nicole Auka, Michael Valle, Bobby D. Cox, Peter D. Wilkerson, Tracey Dawson Cruz, Joseph E. Reiner, Sarah J. Seashols-Williams, Daniel Rittschof.
A single focus optical tweezer is formed when a laser beam is launched through a high numerical aperture immersion objective. This objective focuses the beam down to a diffraction-limited spot, which creates an optical trap where cells suspended in aqueous solutions can be held fixed. Spermatozoa, an often probative cell type in forensic investigations, can be captured inside this optical trap and dragged one by one across millimeter-length distances in order to create a cluster of cells which can be subsequently drawn up into a capillary for collection. Sperm cells are then ejected onto a sterile cover slip, counted, and transferred to a tube for DNA analysis workflow. The objective of this research was to optimize sperm cell collection for maximum DNA yield, and to determine the number of trapped sperm cells necessary to produce a full STR profile. A varying number of sperm cells from both a single-source semen sample and a mock sexual assault sample were isolated utilizing optical tweezers and processed using conventional STR analysis methods. Results demonstrated that approximately 50 trapped spermatozoa were required to obtain a consistently full DNA profile. A complete, single-source DNA profile was also achieved by isolating sperm cells via optical trapping from a mixture of sperm and vaginal epithelial cells. Based on these results, optical tweezers are a viable option for forensic applications such as separation of mixed populations of cells in forensic evidence.
Evidence samples containing biological fluids are frequently encountered in forensic casework, especially with sexual assault cases. Evidence of this nature has the potential to contain a mixed DNA profile, where the combined biological contributions from multiple donors are comingled . Unfortunately, differentiating DNA profiles during downstream mixture interpretation is not a task that is executed with 100% certainty or consistency [2–4]. Thus, for forensic investigations, eliminating mixtures altogether is preferred in order to prevent the problems that accompany interpretation of such data.
Initially, sperm cells were tweezed from neat semen samples. Predictably, as the number of extracted sperm cells increased, the total DNA yield (Fig 2A) and percentage of expected STR alleles from the donor (Fig 2B) also increased. Complete or near complete STR profiles were consistently obtained when ≥50 sperm cells were isolated using this method. Mock sexual assault mixtures were then generated using diluted vaginal epithelial cells and sperm cells of 3 unique pairs of donors, and sperm cells were captured and transferred using the same trapping method (Fig 3). In an effort to evaluate cell loss during the transfer process (after trapping), percent recovery was examined. Recovery of the sperm cells from droplet to coverslip was high, ranging from 58–140% (Table 1). However, it should be noted that in one case (Sample 3, replicate C), additional sperm cells were inadvertently pulled into the capillary from the surrounding region of the droplet, resulting in more sperm collected than intended. Nonetheless, using the trapping and cell transfer method described herein, this data suggests that an approximate 15% cell loss should be anticipated going forward.
The objectives of this study were to optimize sperm cell collection for maximum DNA yield and to determine the number of tweezed sperm cells necessary to produce a complete STR profile using conventional DNA analysis methods. Additionally, we sought to explore how effective optical tweezers could be in separating sperm cells from a mixed sperm:epithelial sample such as is commonly seen in sexual assault evidence.