Modified differential lysis for sexual assault samples using a combined enzymatic and alkaline approach.

Sexual assault sample processing, despite recent funding and research efforts, remains time-consuming, labourious, and inefficient. These limitations, combined with the prevalence of sexual assaults, have prompted the need to develop a cheaper, quicker, and more robust method for separating victim and perpetrator contributions within sexual assault evidence so that analysts can keep pace with submissions and cases can be resolved in a timely manner. Thus, this study examined the use of a combined enzymatic and alkaline approach for differential cell lysis-with the goal of developing a quick, cheap, and more efficient DNA isolation method. Quantification results for this assay revealed that (72.0 ± 18.3)%, (15.8 ± 14.2)%, and (29.5 ± 23.7)% of total DNA were retained in sperm fractions for neat semen, neat vaginal, and semen-vaginal mixture eluates, respectively. Short tandem repeat (STR) analysis of mixture samples processed with this technique exhibited sperm fraction DNA profiles with mean male-to-female ratios of 1.74:1, which was a 3.01 ± 2.30-fold improvement in male-to-female ratios and led to the recovery of 5.90 ± 7.80 unshared male contributor alleles in sperm fractions that were otherwise undetected in unseparated controls. Overall, this study presented a modified differential lysis approach using prepGEM™ and sodium hydroxide treatments that can accomplish cell elution and fractional lysis within 25 min. Future studies should investigate alternative "non-sperm" cell lysis methods to enhance lysis efficiency and minimize the potential for inhibition, as well as the optimization and automation of this technique. Key points: Traditional sexual assault sample processing methods are time-consuming and inefficient.This modified differential lysis method produces lysates with sufficient DNA yield and quality.A combined technique using enzymatic and alkaline lysis can accomplish fractional separation.Lysis with prepGEM and NaOH absent purification is compatible with downstream processes.

Alternative direct-to-amplification cell lysis techniques for forensically relevant non-sperm cells.

While efforts have been made to reduce the pervasive backlog of sexual assault evidence collection kits, the actual laboratory process remains very time-consuming due to the requirement of a differential lysis step before DNA purification, as well as intricate mixture analysis towards the end of the DNA workflow. Recently, an alternative, direct-to-amplification sperm lysis method (using 1 M NaOH) was identified. However, a direct cell lysis method for non-sperm cells has not been identified yet. Thus, the primary objective of this work was to find an alternative method that is quick, inexpensive, and does not require multiple purification steps for the lysis of non-sperm cells in sexual assault samples. In this study, vaginal swab samples were lysed with the control method, prepGEM™, as well as six alternative reagents: alkaline buffer with 25-200 mM NaOH, high-salt stain extraction buffer, modified radioimmunoprecipitation assay (RIPA) buffer, mammalian protein extraction reagent (M-PER™), digitonin buffer, and urea/thiourea buffer. Quantification using Quantifiler® Trio of vaginal and semen lysates revealed that the alkaline (25 mM NaOH) and M-PER™ methods were efficient for the lysis of vaginal epithelial cells without substantial sperm cell lysis. Following quantification, analysis of STR profiles from vaginal lysates revealed that the M-PER™ method showed promising results across all metrics examined, including the percentage of detected STR alleles, mean peak heights, peak height ratio, and interlocus balance. Thus, this method was recommended as an alternative to the traditional differential lysis method for non-sperm cells given its ability to produce amplification-ready lysates without any DNA purification step.

DNA Purification from Bloodstains and Buccal Cells/Saliva on FTA® Cards.

FTA® cards enable efficient, long-term storage of blood and buccal cells/saliva samples for future forensic DNA analysis; these are typically collected as known reference samples, as opposed to evidentiary, crime scene samples. Upon contact with the FTA® card, cells are lysed and the DNA is immobilized. Different FTA® cards are available and have been specially formulated based on sample type: bloodstains are added to the traditional FTA® Card, while colorless sources (e.g., buccal cells/saliva) are added to the FTA® Indicating Card. The main difference between these cards is the presence of a pink dye embedded in the indicating cards that becomes white when exposed to colorless fluids, like saliva; this aids in location confirmation of the stain for future sampling. Although DNA can be eluted/extracted from FTA® punches using various methods or, alternatively, direct STR amplification from unpurified punches can be performed, the protocol herein describes a simple purification method for bloodstained punches from FTA® Cards as well as buccal/saliva-stained punches from FTA® Indicating Cards. Following this purification, STR amplification can be performed via the "punch-in" method.

Alternative direct-to-amplification sperm cell lysis techniques for sexual assault sample processing.

The prevalence of sexual assault cases and increasingly sensitive DNA analysis methods have resulted in sexual assault kit backlogs in the United States. Although traditional DNA extraction and purification utilizing detergents, proteinase K, and DTT have been the primary technique for lysing sperm cell fractions from these samples, it is labor-intensive and inefficient regarding time and sperm DNA recovery - hindering the ability of forensic analysts to keep pace with evidence submissions. Thus, this study examined seven alternative sperm cell lysis techniques to develop a method that could efficiently lyse sperm and consistently generate high-quality profiles while also reducing time, labor, and cost requirements. Microscopic examination of lysates indicated only Casework Direct and alkaline techniques could lyse all spermatozoa within samples, while quantification results demonstrated all methods performed comparably to the control method of forensicGEM™ Sperm (p > 0.06). Amplification with 0.25 ng DNA revealed that unpurified lysates from Casework Direct, alkaline, and NP-40 techniques produced DNA profiles with acceptable mean STR peak heights and interlocus balance, both of which were similar to or better than the control. Overall, this study demonstrated the ability of Casework Direct, alkaline, and NP-40 methods to efficiently lyse spermatozoa and provide high-quality STR profiles despite the absence of a purification step. Ultimately, based on the data reported herein, alkaline lysis is the recommended alternative sperm lysis approach given its ability to generate high-quality profiles, save time, and decrease the cost per reaction when compared to traditional sperm cell lysis methods.

Comparison of DNA typing success in compromised blood and touch samples based on sampling swab composition.

Sample collection at the crime scene can introduce variations in DNA recovery based upon the substrate from which a sample is collected, the material of the collection device used, or the storage conditions after collection. There are many factors during this process that can degrade the sample during drying and storage, and before DNA extraction can be performed. The purpose of this study was to evaluate and compare the performance of standard cotton swab collection with the Bode BioSafe® swab, which includes both a desiccant at the swab head and proprietary compounds to prevent degradation of the sample during sample collection and preservation. Blood and touch DNA samples were collected from porous and nonporous substrates and stored at elevated temperatures to simulate accelerated time. DNA quantification and STR profile data were used to assess the performance of the swabs. BioSafe® swab collection resulted in similar DNA yields from blood samples and significantly higher DNA yields from touch samples when compared to collection with cotton swabs. BioSafe® swabs also resulted in higher DNA integrity during long-term storage, increased STR profile success and improved retention of low-level contributor alleles.

The effects of dithiothreitol (DTT) on fluorescent qPCR dyes.

DNA extractions of semen samples commonly utilize dithiothreitol (DTT) to reduce and disrupt disulfide bonds. Although traditional extraction techniques remove DTT before downstream analyses, the forensic DNA community has recently explored Y-screening, direct amplification, and direct cell lysis assays that omit purification but employ reducing agents to lyse spermatozoa. This study examined the impact of residual DTT on downstream processes involving fluorescent dyes. Quantification using Investigator® Quantiplex HYres revealed a significant increase in the male DNA yield (p = 0.00056) and a >150,000,000-fold increase in the male:human DNA ratio when DTT remained in extracts versus when it was filtered out using a traditional purification method. When DTT was present with Quantifiler™ Trio, the true mean DNA yield for the large autosomal target significantly increased (p = 0.038) and the average reported DNA yields increased 1.1-fold, >9.5-fold, and 1.3-fold for the small autosomal, large autosomal, and male targets, respectively. DTT-spiked DNA standards from both kits were impacted similarly to samples with residual DTT, demonstrating that observed effects were related to DTT and not the extraction method. This study corroborates other reports that DTT adversely affects multiple dyes (e.g., Cy5, Quasar 670, SYBR Green I, TMR, and Mustang Purple® ). Overall, DTT causes inaccurate quantities and, consequently, inaccurate calculated male:female ratios when used in conjunction with these kits. Thus, implementation of newer direct-to-PCR assays incorporating DTT should either be avoided or used only with carefully evaluated, compatible dyes.