Automation of the Differential Digestion Process of Sexual Assault Evidence,

Abstract

Sexual assault evidence samples require the use of a specific process known as a differential digestion to separate sperm from nonsperm cells prior to DNA extraction. An automated differential digestion process was developed using a selective degradation technique, which uses DNase I to digest the remaining nonsperm DNA in the sperm fraction. The use of DNase on pristine samples, as well as aged and degraded samples, was assessed to ensure that the quantity and quality of the sperm DNA were not compromised or adversely affected. Samples processed using the selective degradation technique yielded comparable DNA yield and DNA typing data to the conventional differential digestion process. The automated process utilized 96-well plates for high throughput and incorporated microscope slide preparations for confirmation of sperm. It reduced processing time by about sixfold and was paramount in the elimination of the Oakland Police Department Criminalistics Laboratory's sexual assault kit backlog.

Recent trends and developments in forensic DNA extraction

“…The significance of reducing female DNA carryover towards enhancing the potential for detecting a male STR profile in the spermic fraction has been demonstrated with the use of the Erase Sperm Isolation Kit which incorporates a DNase I treatment step (Garvin et al, 2012; Klein & Buoncristiani, 2017). Although DNase treatment has been demonstrated to be effective in degrading non‐spermic DNA from epithelial cell carryover in spermic fractions, some studies suggest that the remnant DNase activity may cause degradation and reduction in male DNA recovery (Garvin et al, 2009; Martinez, Nori, Dimsoski, & McCord, 2017; Nori & McCord, 2015; H. Wong & Mihalovich, 2019). Current DE protocols can be modified accordingly to incorporate DNase treatment (Hudlow & Buoncristiani, 2012; Zhao et al, 2016) with minimal impact on automation (Clark et al, 2019; H. Wong & Mihalovich, 2019).…”

“…Although DNase treatment has been demonstrated to be effective in degrading non‐spermic DNA from epithelial cell carryover in spermic fractions, some studies suggest that the remnant DNase activity may cause degradation and reduction in male DNA recovery (Garvin et al, 2009; Martinez, Nori, Dimsoski, & McCord, 2017; Nori & McCord, 2015; H. Wong & Mihalovich, 2019). Current DE protocols can be modified accordingly to incorporate DNase treatment (Hudlow & Buoncristiani, 2012; Zhao et al, 2016) with minimal impact on automation (Clark et al, 2019; H. Wong & Mihalovich, 2019). Prior to adopting such an approach, forensic DNA laboratories are advised to consider whether their respective legal systems require the detection and reporting of the female victim's DNA profile to demonstrate the source of the intimate sample.…”

“…A more recent semi‐automated DE method has been reported involving the use of the VERSA 1100 liquid handler (H. Wong & Mihalovich, 2019). The system requires only four manual steps to allow for centrifugations, slides and tubes removal, sample agitation, and reagent loading.…”

Developmental validation of an efficient differential separation method incorporating the i‐sep®DL spin column with high sperm DNA recovery for the processing of sexual assault samples

“…Automation of the differential process has the potential to greatly reduce cross-contamination risks from manual handling and reduce inter-operator variability. However, the results from studies that have investigated partial [10,11] and full automation [13] of differential methods reported that DNA yield and variability were not sufficiently improved compared with the manual pellet-based differential method. In this study, the i-sep ® differential method eliminates the key offender to variability, the physical agita- (2013) found that different lysis conditions of non-sperm fraction such as concentration of proteinase K and SDS have little effect on premature lysis of sperm cells [22].…”

“…Since its inception, many laboratories have introduced alterations in an attempt to improve this pellet-based differential separation and extraction method (i.e., enhance cell release from sampling substrates; increase capture of sperm cell DNA; and reduce nonsperm carryover in the final sperm fraction) [2][3][4][5][6][7][8] or to automate components of the differential separation process [9][10][11]. Although these modifications have been beneficial, the differential separation process remains time-consuming and laborious, involving many manual manipulations of the sample substrate and the sperm pellet.…”