Forensics
by Allison Holt, Sheri Olson, Michele Marfori and Denise Yong Ning Oh
A DNA-Based Screening Assay to Streamline Sexual Assault Sample Processing
S
exual assault kit (SAK) samples account for a significant por- tion of backlogged cases in forensic laboratories. It can take 4–6 hours to screen SAK evidence, and there may be wide varia- tion among the type and age of samples collected, which can require further analysis time.
If sperm is detected, the sample is processed using diff erential extraction to separate the suspect sperm fraction from the victim’s epithelial cells in order to improve DNA-based identifi cation downstream. However, many SAKs often do not yield any detectable male DNA when analyzed downstream by autosomal and Y short-tandem repeat (STR) amplifi cation.
As an alternative to time-consuming, labor-intensive diff erential extrac- tion, the DNA Y-Screen assay (Applied Biosystems, Foster City, Calif. [part of Thermo Fisher Scientifi c]) assesses swab evidence from SAKs to rap- idly detect the presence of a male contributor. In conjunction with other presumptive serological and microscopic slide screening methods, the assay allows the forensic laboratory to confi rm serology results, and is an eff ective tool for detecting male/sperm DNA when slide-screening results are questionable.
Y-Screen assay The assay, which has been used in previous DNA extraction protocols,
including differential extraction,1–3 utilizes NaOH to rapidly lyse cells (in-
cluding sperm) and the Quantifiler Trio DNA quantification kit (Applied Biosystems) to detect the presence or absence of male DNA prior to stan- dard SAK sample processing. A 7500 Real-Time PCR instrument (Applied Biosystems) with HID Real-Time PCR analysis software v1.2 is used to run the assay. The kit contains primers and probes for several targets—two autosomal human-specific targets (80 and 214 bp), one male human- specific target (75 bp) and an internal positive control target (IPC) (130 bp). The ratio of these quantitative autosomal targets provides a qualitative determination of the level of degradation present in the sample. The IPC in each reaction contains a synthetic DNA template, offers positive confirmation that all assay components are functioning as expected, and is a useful indicator of inhibition present in the sample. All of the human targets in the Quantifiler Trio kit are multicopy targets, which provides for subpicogram-level detection of both human autoso- mal and male DNA in one assay.
Y-Screen assaying comprises the following steps: 1. Cut ~1/10 from each cotton swab sample type.
Table 1 – Y-Screen assay results and corresponding STR results with YFiler and/or Identifi ler Plus kits
Figure 1 – Identifi ler Plus results for purifi ed DNA from a buccal sample from a female donor containing 50 µL saliva from a male donor.
2. Place swab into PrepFiler LySep column assembly (column and sample tube) (Applied Biosystems). Add 100 µL 1N NaOH to the column containing the swab cutting and place in a heat block preheated to 80 °C. Shaking at ~750 rpm is recommended. Incubate at 80 °C for 10 minutes.
AMERICAN LABORATORY • 42 • AUGUST 2015
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