Application of Microfluidic Technology for Single Sperm Cell Isolation and STR Typing from Multi-Donor Sperm Mixture

Mixed DNA evidence is characterized by a significant difficulty in differentiating contributor genotypes. Biological evidence recovered from a sexual assault crime scene including the victim's body and clothing can be a mixture of female-male or male-male cells from multiple contributors. Conventional differential extraction methods can be applied to separate sperm cells from a large amount of epithelial cells. However, the recovery rate of sperm cells may be largely reduced during extraction and it is ineffective for separating sperm cells from multiple contributors. New developments in microfluidic technologies allow efficient and high throughput approaches for single-cell analysis and have forensic potential. For one such technology, the recent introduction of a size-selective chip in a smaller size range provides an opportunity to capture and separate single spermatozoa, which can be used for resolving mixtures encountered in multiple perpetrator sexual assault (MPSA) cases. The aim of this study was to establish a feasible protocol for the sperm cell preparation in C1 Single-Cell Auto Prep System and apply its microfluidic technology to differentiate a sperm cell mixture from multiple donors and obtain consistent STR profiles of each individual.;In order to establish the protocol, several parameters were examined, including centrifugation speed, wash buffer, centrifugation time, removal of round cells, and fluorescent staining. Additionally, Tris(2-carboxyethyl) phosphine, or TCEP, was experimented to increase the DNA yield in whole-genome amplification of multiple displacement amplification (WGA-MDA). The results indicated C1 Single-Cell Auto Prep System could capture spermatozoa/spermatogenic cells successfully yet WGA-MDA provided limited efficiency for downstream STR analysis. The capture rate was up to 64.6% (n=62). 42 partial STR profiles of the captured cells were obtained and the maximal marker call rate was 93.8% (n=15). In addition, on the off-chip tests of MDA, TCEP yielded approximately 1.8 fold greater amounts of DNA from 1 Million/mL sperm cell samples than DTT.;Using the optimized protocol, we succeeded in capturing single sperm cells and resolving a sperm cell mixture from two individuals using STR analysis. Our optimized procedure for cell isolation of spermatozoa using microfluidics has broad applications in genetics and in vitro fertilization (IVF). However, significant modifications are required in order to be applicable to forensic analysis. Several approaches may be applied in a future study in order to both increase the capture rate and improve the efficiency in downstream analysis, such as cell sorting, the implementation of a different amplification method, and the application of other tools in downstream analysis. Although further studies are needed, based on the success of STR analysis from single sperm cells in our study, the C1 System shows promise for resolving mixtures often encountered in sexual assault cases.