DNA-BASED SCREENING continued


Table 2 –Y-Screen assay results and corresponding STR results with YFiler and/or Identifi ler Plus kits


Discussion The protocol described supports only swab samples and was not tested


on other substrates by the Utah Bureau of Forensic Services (UBFS). All swabs are known to have variable amounts of cellular material and free DNA across the swab itself, which is also a problem for serological and slide screening methods. For this reason, the assay must be validated with a uniform cutting procedure to help reduce the eff ects of sample heterogeneity. Some users have taken multiple cuttings of the same swab to improve yields and accuracy.


While very low quantifi cation results with the Y-Screen assay generally correlate to low quantifi cation results post-extraction, this is not always the case for all samples. Therefore, it is not recommended to make direct correlations between the pre-extraction (Y-Screen) and post-extraction quantifi cation values. Any inhibitors present in the Y-Screen lysate from samples may impact the IPC and degradation index results, but are typi- cally removed through standard diff erential extraction cleanup methods. Quantifi ler Trio using HID Real-Time PCR analysis software v1.2 provides clear warning signs of sample and reaction quality when the Y-Screen results are diffi cult to interpret, information that is not available with traditional serological assays.


Conclusion Due to high backlogs of SAK samples, laboratories must make critical deci-


sions about which samples to process. Sample screening using traditional serological assays and microscopic sperm slide searches are done early in the workfl ow. The DNA-based Y-Screen assay, using Quantifi ler Trio and HID Real-Time PCR analysis software v1.2, enhances these screening methods with intelligent sample quantity and quality interrogation tools. Moreover, correlation between pre-extraction quantifi cation results and downstream STR results has been demonstrated.


Y-Screen assay with mock casework samples A set of 20 mock casework samples was created with a range of bodily


fl uids including sperm (seminal fl uid, SF) diluted in TE-4, blood, saliva and touch (see Table 2). Mixtures 1–4 were buccal swabs from a female donor with sperm diluted as described and 50 µL of each dilution of sperm added. Mixtures 6 and 8–10 were prepared with 40 µL whole female blood and an equal amount of the listed seminal fl uid dilution. Mixture 7 was similarly prepared using 60 µL of female blood and 20 µL of a diluted seminal fl uid. Mixture 5 was a previously prepared training sample for which the volumes were not recorded. Samples Touch 2 and Touch 4 were neat male saliva on a swab rubbed on a target arm and then collected with a fresh swab. Samples Touch 1 and 3 were swabbings collected from an individual’s arm after being grabbed on the arm by another participant.


As with the previous dilution series data, in all cases the Y-Screen assay was predictive of downstream results; a positive male quantifi cation result with Y-Screen correlated with a positive male quantifi cation result post- extraction. For the three samples with a zero male quantifi cation result, no samples provided useful STR information. For Mixture 1, the sample was not amplifi ed because current laboratory practice for casework is to stop at quantifi cation if the result is zero.


References 1. Hudlow, W.R. and Buoncristiani, M.R. Development of a rapid, 96-well alkaline based diff erential DNA extraction method for sexual assault evidence. Forensic Sci. Int. Genet. 2012, 6(1), 1–16.


2. Rudbeck, L. and Dissing, J. Rapid, simple alkaline extraction of human genomic DNA from whole blood, buccal epithelial cells, semen and forensic stains for PCR. Biotechniques 1998, 25(4), 588–92.


3. Truett, G.E.; Heeger, P. et al. Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT). Biotech- niques 2000, 29(1), 52–4.


Allison Holt is staff scientist, and Sheri Olson is senior product man- ager, Thermo Fisher Scientific, 180 Oyster Point Blvd. East, South San Francisco, Calif. 94090, U.S.A.; tel.: 650-876-1949; e-mail: Allison.Holt@thermofisher.com; www.thermofisher.com. Michele Marfori is forensic scientist quality manager, and Denise Yong Ning Oh is forensic scientist 1, Utah Department of Public Safety, Salt Lake City, Utah, U.S.A. The data presented were generated by the Utah Bureau of Forensic Services [UBFS]. Quantifi ler Trio, 7500 Real-Time PCR instrument, HID Real-Time PCR software v1.2, PrepFiler LySep, AmpF1STR YFiler and AmpF1STR Identifi ler Plus are for forensic or paternity use only, unless stated otherwise on indi- vidual product labeling.


AMERICAN LABORATORY • 44 • AUGUST 2015


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