Description
Sexual assault samples are commonly comprised of a mixture of non-sperm and sperm cells from multiple individuals. EZ2 DNA Investigator Sep&Prep Kit is based on the principle of differential extraction, which uses the different properties of sperm cells and all other cells that occur in sexual assault samples. This enables the generation of individual profiles of the perpetrator from whom the sperm cells originated. EZ2 DNA Investigator Sep&Prep Kit contains all required reagents and labware to separate the non-sperm fraction from the sperm fraction and automate the purification of genomic sperm-derived DNA on the EZ2 Connect Fx instrument. Magnetic-particle technology provides high-quality DNA from the sperm fraction that is suitable for direct use in downstream applications. EZ2 Connect Fx performs all steps of the sample separation and preparation procedure of up to 24 samples in a single run of ca. 70 minutes. The automated workflow provides reduced hands-on time, high ease-of-use, increased throughput and highly reproducible results in a short run time in order to meet the increased demand for the timely analysis of sexual assault evidence kits and the processing of backlogs. For purification of the non-sperm fractions (NSF), we recommend the use of EZ1&2 DNA Investigator Kit which can either be done directly afterwards on the same EZ2 Connect Fx instrument or at a later timepoint. EZ2 DNA Investigator Sep&Prep Kit meets ISO 18385 requirements.
EZ2 DNA Investigator Sep&Prep Kit is characterized by improved sperm DNA recovery and precise separation between sperm-derived DNA and DNA coming from non-sperm cells (see figure “ Ratio of human: male DNA in the sperm fractions ” and figure “ Precise separation even with challenging samples ”). It is highly effective in handling challenging samples with high female DNA and low male DNA amounts and is capable of producing single-person profiles or mixed profiles with a distinct male major component which often result in mixed STR profiles using other methods (see figure “ Distinct profiles from mixed samples ”).
EZ2 Connect Fx provides fully automated separation of the non-sperm fraction from the sperm fraction and preparation of sperm-derived genomic DNA using silica-coated magnetic particles. Magnetic-particle technology eliminates tedious centrifugation steps, and the easy-to-use instrument allows purification of 1–24 samples in a single run. The automatic sensor-controlled lock of the instrument door and the use of sealed reagent cartridges reduce the risk of environmental contamination. Furthermore, the simplified and streamlined workflow further reduces the risk of processing errors and user-to-user variability, which may happen with manual differential extraction according to Gill et., 1975 (1). (1): Gill P, Jeffreys AJ, Werrett DJ (1985) Forensic application of DNA fingerprints. Nature 318:577–579 Features of EZ2 DNA Investigator Sep&Prep Kit Feature Details Reactions per kit 48 Sample type Sexual assault samples, e.g., post-coital swabs, vaginal or buccal swabs with semen, sperms mixed with different cell types, ejaculate on fabrics, etc. Processing Automated Compatibility EZ2 Connect Fx Technology Magnetic particles Sample volume 500 µL Elution volumes 50 µL, 100 µL Time per instrument run ca. 70 min incl. removal of NSF*, wash steps, DNA preparation, ProtK inactivation Total time PCR-ready extracts from sperm fraction in around 2.5 hours Output Ready-to-use sperm-derived DNA, non-sperm fraction † Downstream analysis qPCR, dPCR, STR, NGS *NSF = Non-sperm fraction. † The non-sperm fraction can be stored or purified with EZ1&2 DNA Investigator Kit (48) on the same instrument.
Mixed sexual assault samples consisting of non-sperm and sperm cells are pre-treated (first lysis), centrifuged and loaded onto the EZ2 Connect Fx instrument. After removal of the supernatant (non-sperm fraction, NSF), EZ2 Connect Fx performs a number of purification steps. Sperm-derived genomic DNA is eluted and collected in the Elution Tube in rack position D. It is ready to use for any downstream application (qPCR, STR, NGS). The non-sperm fractions is available in the Sample Tube in rack position A. These can either be immediately purified by using EZ1&2 DNA Investigator Kit on the same instrument (without changing the rack position) or removed and stored for purification at a later time point (see "Workflow overview ”). Optional protocols for spermatozoa staining and/or chromatographic immunoassay (e.g., PSA) can be combined with the Sep&Prep workflow.
